Methods relating to pluripotent cells

ABSTRACT

The technology described herein relates to methods, assays, and compositions relating to causing a cell to assume a more pluripotent state, e.g. without introducing foreign genetic material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/955,362 filed Mar. 19, 2014, 61/955,358filed Mar. 19, 2014, and 62/043,042 filed Aug. 28, 2014, the contents ofwhich are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The technology described herein relates to the production of pluripotentcells.

BACKGROUND

Current methods of obtaining pluripotent cells rely primarily upontissues of limited availability (e.g. embryonic tissue or cord blood) orthe addition of reprogramming factors (Hanna, J. et al. Cell 2008 133,250-264; Hockemeyer, D. et al. Cell stem cell 2008 3, 346-353; Kim, D etal. Cell stem cell 2009 4, 472-476; Kim, J B Nature 2009 461, 649-643;Okabe, M. et al. Blood 2009 114, 1764-1767), which involves introductionof exogenous nucleic acids. Methods of readily producing stem cells,particularly autologous stem cells, without the complications introducedby the addition of exogenous reprogramming factors, would accelerateresearch into cellular differentiation and the development of stem-cellbased therapies. While it is hypothesized that damage to cells as aresult of exposure to irritants, such as burns, chemical injury, traumaand radiation, may alter normal somatic cells to become cancer cells,there is no direct evidence that healthy adult somatic cells can beconverted to other states without the specific manipulation ofreprogramming factors.

Previously, researchers have reported finding “adult stem cells” inadult tissues (Reynolds, B. A. & Weiss, S. Science 1992 255, 1707-1710;Megeney, L. A. et al., Genes & development 1996 10, 1173-1183; Caplan,A. I. Journal of orthopaedic research 1991 9, 641-650; Lavker, R. M. &Sun, T. T. The Journal of investigative dermatology 1983 81, 121s-127s).Such reports remain controversial. For example, researchers looking forcells expressing the stem cell marker Oct4 failed to findOct4-expressing cells in adult bone marrow in normal homeostasis,(Lengner, C. J. et al. Cell Cycle 2008 7, 725-728; Berg, J. S. &Goodell, M. A. Cell stem cell 2007 1, 359-360), while others report theability to isolate Oct4-expressing cells from different adult tissues(Jiang, Y. et al. Nature 2010 418, 41-49; D'Ippolito, G. et al. Journalof cell science 2004 117, 2971-2981; Johnson, J. et al. Cell 2005 122,303-315; Kucia, M. et al. Leukemia 2006 20, 857-869; Kuroda, Y. et al.PNAS 2011 107, 8639-8643; Obokata, H. et al. Tissue engineering. 2011Part A 17, 607-615; Rahnemai-Azar, A. et al. Cytotherapy 2011 13,179-192; Huang, Y. et al. Transplantation 2010 89, 677-685; Zuba-Surma,E. K. et al. Journal of cellular and molecular medicine 2011 15,1319-1328; Paczkowska, E. et al. Annals of transplantation 2011 16,59-71). It has been hypothesized that these cells represent either apopulation of adult stem cells or are merely an artifact of thetechniques being used. In either case, they remain rare and do notrepresent an adequate source of pluripotent cells for research andtherapeutic purposes.

SUMMARY

Described herein are improved methods for generating pluripotent cells,e.g. STAP cells which provide increased efficiency, yield, and/orquality as compared to the methods disclosed in International PatentPublication WO 2013/163296 and Obokata et al. Nature 2014 505:641-647;each of which is incorporated by reference herein. Also described hereinare methods and uses relating to cells generated by the present methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict Oct4 expressing cell generation from CD45 positivesomatic cells. FIG. 1A depicts Oct4-GFP expression of stress treatedcells. Stress-treated cells express Oct4-GFP, while untreated controlsdid not. Magnification of an Oct4-expressing colony is shown in theupper right in the stress-treated group. Scale bar indicates 100 μm.FIG. 1B depicts population analysis of stress-treated cells andnon-stress treated control. A GFP expressing cell population is observedonly in the stress treated group at day 5. FIG. 1C depicts cell-sizeanalysis of CD45 positive cells before and after the stress treatment atday 7. FIG. 1D depicts chronological change of CD45 positive cells afterthe stress treatment.

FIGS. 2A-2B depict characterization of animal callus cells (ACCs). FIG.2A depicts chronological gene expression change of pluripotent markergenes. The messenger RNA levels were normalized to GAPDH. (n=3, theaverage+S.D.) FIG. 2B depicts methylation analysis of Oct4 and Nanogpromoter genes.

FIGS. 3A-3D depict cellular modifications after stress treatment. FIG.3A depicts relative gene expression of stress defense genes during theACCs generation phase. Samples were collected at day 3 and day 7 andcompared with CD45 positive cells. (n=3, the average+S.D.) FIG. 3 Bdepicts total cellular ATP measurement. (n=3, the average+S.D.) FIG. 3Cdepicts ROS measurement. Error bars indicate SD. FIG. 3D depictsrelative gene expression of mtDNA replication factors. (n=3, theaverage+S.D.)

FIGS. 4A-4B depict chimera mouse generation from ACCs. FIG. 4A depicts ascheme of chimera mouse generation. Panel (i) demonstrates that ACs weredissociated into single cells with trypsin or (panel ii) ACs were cutinto small pieces then injected into blastocysts. FIG. 4B depictschimera contribution analysis. Tissues from 9 pups were analyzed byFACS.

FIGS. 5A-5C experiments with ACC-generating conditions. FIG. 5Ademonstrates that CD45 positive cells were exposed to various stressesand Oct4-GFP expression was analyzed by FACS. Percentage of Oct4-GFPexpressing cells in survived cells after stress treatment. (n=3, theaverage+S.D.) FIG. 5B depicts the determination of pH condition. CD45positive cells were exposed to different pH solutions. At 3 days afterstress treatment, Oct4-GFP expression was analyzed by FACS. FIG. 5Cdepicts the determination of culture condition. Stress treated cellswere cultured in various mediums. The number of GFP-expressing ACs wascounted at day 14. (n=3, the average+S.D.)

FIGS. 6A-6B depict ACCs generation from CD45 positive cells derived fromICR mice. FIG. 6A depicts chronological change of CD45 positive cellsafter stress treatment. The expression of E-cadherin and SSEA-1 wasanalyzed by FACS. FIG. 6B demonstrates that Oct4 gene expression ofE-Cadherin/SSEA1 double positive cells was confirmed by RT-PCR. (n=3,the average+S.D.)

FIGS. 7A-7B depict ACC generation from various tissues derived from GOFmice. FIG. 7A depicts the ratio of Oct4-GFP expressing cells afterstress treatment. Somatic cells were isolated from various tissues, andexposed to various stresses. Oct4-GFP expression was analyzed by FACS.FIG. 7B depicts embryonic gene expression of ACCs derived from varioustissues. Gene expressions were normalized by GAPDH. (n=3, theaverage+S.D.)

FIG. 8 depicts relative gene expression of stress defense genes duringthe first 7 days. After stress treatment, cells were collected at day 1,3 and 7, and gene expression was compared with native CD45 positivecells. Blue graphs indicate the gene expressions of heat shock proteins.Green graph indicates DNA repair gene expression. Red graphs indicatethe gene expression of redox genes. Y-axis indicates relative folds ofexpression.

FIG. 9 depicts differentiation of ACCs. The graph depicts a chimeracontribution analysis. Chimera fetuses generated with ACCs derived fromvarious somatic cells were analyzed by FACS. Graph shows the average of5 chimera fetuses at E13.5 to 15.5.

FIG. 10 demonstrates that stress treatment caused reprogramming tosomatic cells via Mesenchymal-Epithelial Transition (MET). Theexpression of MET-related genes is shown in native cells, and in cells 3and 7 days after stress treatment was begun. The y-axis shows %expression, normalized to the level in the sample with the expressionlevel for that gene.

FIG. 11 depicts FACS analysis of cell populations before and afterstress. GFP expression was evident, indicating generation of pluripotentcells, in post-stressed cell populations from each tested tissue type.

FIGS. 12A-12E demonstrate low-pH treatment induced fate conversion incommitted somatic cells. FIG. 12A depicts a schematic the experimentalprotocol. FIG. 12B depicts flow cytometry analysis (Top row:oct3/4::GF^(P+)/CD4⁵⁻; bottom row: non-treated CD4⁵⁺ cells). The y axisis the number of Oct3/4:GFP cells, and the X axis is the number of CD45+cells. Both axes are marked in major units of 0, 100, 1000, and 10,000.FIG. 12C depicts a graph of viable oct3/4::GFP⁺ and oct3/4::GFP− cellsover time in culture. FIG. 12D depicts a graph of cell size ofOct3/4::GFP+ cells (left peak) and CD45+ cells (right peak). FIG. 12Edepicts the results of analysis of genomic rearrangements of tcrβ inisolated oct3/4::GFP⁺ spheres by genomic PCR.

FIGS. 13A-13B demonstrate that low-pH-induced Oct3/4⁺ cells havepluripotency. FIG. 13A depicts a graph of gene expression analysis byqPCR in low-pH-induced oct3/4::GFP⁺ cells on d7 as compared to CD45⁺cells (the series represent, from left to right, oct3/4, nanog, sox2,ecat1, esg1, dax1 and klf4 expression). Samples were collected at day 3and day 7 and compared with CD45 positive cells. (n=3, the average+S.D.)FIG. 13B depicts the results of bisulfate sequencing was of the oct3/4and nanog promoter areas. CD45⁺ cells, with or without additionalculture, displayed heavily methylated patterns at both promoters.

FIGS. 14A-14B demonstrate that STAP cells can be obtained from othertissue sources. FIG. 14A depicts a graph of the rate of production ofoct3/4::GFP⁺ cells at d7 culture for a number of tissues (the seriesrepresent, from left to right, CD45+ cells, bone marrow, brain, lung,muscle, adipose, fibroblasts, liver, and chondrocytes). FIG. 14B depictsa graph of gene expression analysis in oct3/4::GFP⁺ cell clusters (theseries represent, from left to right, the expression o Oct3/4, Nanog,Sox2, Klf4, and Rex1).

FIGS. 15A-15B depict the characterization of STAP cells as pluripotentcells. FIG. 15A depicts a graph of gene expression of ES cell markers inSTAP cells (series represent, from left to right, ES, EpiSC, STAP, andCD45). FIG. 15B depicts a graph of the % of X-chromosomal inactivationin STAP cells.

FIG. 16A depicts a graph of Oct4-GFP expression analyzed by FACS in CD45positive cells exposed to various stresses. Percentage of Oct4-GFPexpressing cells in survived cells after stress treatment. (n=3, theaverage+S.D.) FIG. 16B depicts a graph of determination of pH condition.CD45 positive cells were exposed to different pH solutions. At 3 daysafter stress treatment, Oct4-GFP expression was analyzed by FACS. (n=3,the average+S.D.) FIG. 16C depicts a graph of determination of culturecondition. Stress treated cells were cultured in various mediums. Thenumber of GFP-expressing stress altered cell mass was counted at day 14.(n=3, the average+S.D.)

FIGS. 17A-17B depict SACs generation from CD45 positive cells derivedfrom ICR mice. FIG. 17A depicts the chronological change of CD45positive cells after stress treatment. The expression of E-cadherin andSSEA-1 was analyzed by FACS. FIG. 17B depicts a graph of Oct4 geneexpression of E-Cadherin/SSEA1 double positive cells, confirmed byRT-PCR. (n=3, the average+S.D.)

FIGS. 18A-18B depict SACs generation from various tissues derived fromGOF mice. FIG. 18A depicts a graph of the ratio of Oct4-GFP expressingcells after stress treatment. Somatic cells were isolated from varioustissues, and exposed to various stresses. Oct4-GFP expression wasanalyzed by FACS. Series represent, from left to right, BM, brain, lung,muscle, fat, fibroblast, and liver. FIG. 18B depicts a graph ofembryonic gene expression of SACs derived from various tissues. Geneexpressions were normalized by GAPDH. (n=3, the average+S.D.) Seriesrepresent, from left to right, Oct4, Nanog, Sox2, Klf4, and Ecat1.

FIG. 19 depicts a graph of relative gene expression of stress defensegenes during the first 7 days. After stress treatment, cells werecollected at day 1, 3 and 7, and gene expression was compared withnative CD45 positive cells. Y-axis indicates relative folds ofexpression.

FIG. 20 depicts TCRβ chain rearrangement analyses of SACs and chimericmice derived from SACs from CD45+ cells. 2N chimeric mice #1, #2, #3,#5, #6, #7, #8 and #9 expressed rearranged DNA.

FIG. 21 depicts genotyping analysis of 4N chimeric mice. Genotyping wasperformed to prove that 4N chimeric mice generated with SACs derivedfrom 129/Sv×B6GFP F1 and 4N blastocysts derived from ICR expressed SACs(129/Sv×B6GFP) specific gene.

FIG. 22 demonstrates that STAP cells contribute to both embryonic andplacental tissue in vivo. The graph depicts the ratio of fetuses inwhich injected cells contributed only to the embryonic portion and alsoto placental and yolk sac tissues.

FIGS. 23A-23C demonstrate that FGF4 treatment induces sometrophoblast-lineage character in STAP cells. FIG. 23A depicts aschematic of FGF4-treatment to induce TS-like (F4I) cells from STAPcells. FIG. 23B depicts a graph of qPCR analysis of marker expression.FIG. 23C depicts a graph of quantification of placental contribution byFACS analysis. Unlike F4I cells, ES cells did not contribute toplacental tissues at a detectable level.

FIGS. 24A-24D demonstrate that ES cell-like stem cells can be derivedfrom STAP cells. FIG. 24A depicts a schematic of induction of stem celllines from STAP cells. FIG. 24B depicts a graph demonstrating robustgrowth of STAP-S cells in maintenance culture over 120 days. Similarresults were obtained with 16 independent lines. In contrast, parentalSTAP cells decreased in number quickly. FIG. 24C depicts a graph of qPCRanalysis of marker gene expression. ES and STAP-S cells expressedpluripotency-related genes that were not expressed in CD45⁺ cells. FIG.24D depicts a schematic representation of DNA methylation study bybisulfate sequencing.

FIGS. 25A-25B demonstrate that STAP stem cells are pluripotent andcompatible with germ line transmission and tetraploid complementation.FIG. 25A depicts a graph of the contribution of STAPS cells to varioustissues in chimera mice in blastocyst injection assays (2N). FIG. 25Bdepicts a graph of the contribution to placental tissues. Unlikeparental STAP cells and TS cells, STAPS cells no more retained theability for placental contributions. Three independent lines were testedand all showed substantial contributions to the embryonic portions.

FIG. 26 demonstrates the pluripotency induced by the acid wash.

FIG. 27 depicts, top: Mechanical hyperalgesia, indicated by the drop inpaw withdrawal threshold after capsaicin injection, is reduced in ratstreated with intrathecal SSP-SAP. Subsequent graphs show the response at10 min post-capsaicin, when the largest difference occurs. Bottom: Fiveweeks after spinal stem cells were implanted the capsaicin-inducedhyperalgesia is restored.

FIG. 28 depicts tactile (top) and thermal (bottom) responses aftercapsaicin injections into the paw in rats first injected i.t. withSSP-SAP, that greatly reduces the hyperalgesic state (cf. FIG. 27), andthen treated with stem cells, lumbar i.t. injection. “Naïve Response”shows the hyperalgesic response to capsaicin before any manipulations.“BL1” is the baseline response before a capsaicin injection in rats thathad been treated 2 weeks previously with either SSP-SAP or the inactiveBlank-SAP. “BL2” is the baseline response, without capsaicin injection,1-2 days after the Stem cell delivery. Note the ability of the stem cellimplant to return the hyperalgesic response of SSP-SAP-treated rats tothat of Naïve rats and of Blank-SAP-treated controls.

FIG. 29 demonstrates that the potency of a specific antagonist of theNK1-R is increased in rats where capsaicin sensitivity has been restoredby stem cell implants. The IC50 of L-733,060 is ˜0.3 mM (30 uL i.t.injection) for both modes of hyperalgesia in naïve rats (O, □; leftpanel; and in those rats that received Blank-SAP followed by stem cells,not shown), whereas in the stem cell-restored rats (right panel) theIC50 is ˜30 uM for tactile hyperalgesia (▪) and ˜5 uM for thermalhyperalgesia ().

DETAILED DESCRIPTION

Aspects of the technology described herein relate to the production orgeneration of pluripotent cells from cells. The aspects of thetechnology described herein are based upon the inventors' discovery thatstress can induce the production of pluripotent stem cells from cellswithout the need to introduce an exogenous gene, a transcript, aprotein, a nuclear component or cytoplasm to the cell, or without theneed of cell fusion. In some embodiments, the stress induces a reductionin the amount of cytoplasm and/or mitochondria in a cell; triggering adedifferentiation process and resulting in pluripotent cells. In someembodiments, the stress causes a disruption of the cell membrane, e.g.in at least 10% of the cells exposed to the stress. These pluripotentcells are characterized by one or more of, the ability to differentiateinto each of the three germ layers (in vitro and/or in vivo), thegeneration of teratoma-like cell masses in vivo, and the ability togenerate viable embryos and/or chimeric mice.

Described herein are experiments demonstrating that treatment of cellswith certain environmental stresses, including, but not limited tostresses which reduce the amount of cytoplasm and/or mitochondria in thecell, can reduce mitochondrial activity, demethylate regions of thegenome associated with dedifferentiation, cause the cells to displaymarkers of known dedifferentiation pathways. Accordingly, in someembodiments, provided herein are methods of generating pluripotent cellsfrom cells, the methods comprising removing at least about 40% of thecytoplasm and/or mitochondria from a cell, and selecting pluripotency orcells exhibiting pluripotency markers, wherein the cell is not presentin a tissue. Also described herein are other stress treatments that cangenerate pluripotent cells from cells.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), and The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9).Definitions of common terms in molecular biology can also be found inBenjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009(ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology andBiotechnology a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols inProtein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); Current Protocols in Cell Biology (CPCB) (Juan S.Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture ofAnimal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher:Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods inCell Biology, Vol. 57, Jennie P. Mather and David Barnes editors,Academic Press, 1st edition, 1998) which are all incorporated byreference herein in their entireties.

The terms “decrease,” “reduce,” “reduced”, and “reduction” are all usedherein generally to mean a decrease by a statistically significantamount relative to a reference. However, for avoidance of doubt,“reduce,” “reduction”, or “decrease” typically means a decrease by atleast 10% as compared to the absence of a given treatment and caninclude, for example, a decrease by at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, up to and including, forexample, the complete absence of the given entity or parameter ascompared to the absence of a given treatment, or any decrease between10-99% as compared to the absence of a given treatment.

The terms “increased”, “increase”, or “enhance” are all used herein togenerally mean an increase by a statically significant amount; for theavoidance of any doubt, the terms “increased”, “increase”, or “enhance”means an increase of at least 10% as compared to a reference level, forexample an increase of at least about 20%, or at least about 30%, or atleast about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” when used in reference to a disease, disorder or medicalcondition, refer to therapeutic treatments for a condition, wherein theobject is to reverse, alleviate, ameliorate, inhibit, slow down or stopthe progression or severity of a symptom or condition. The term“treating” includes reducing or alleviating at least one adverse effector symptom of a condition. Treatment is generally “effective” if one ormore symptoms or clinical markers are reduced. Alternatively, treatmentis “effective” if the progression of a condition is reduced or halted.That is, “treatment” includes not just the improvement of symptoms ormarkers, but also a cessation or at least slowing of progress orworsening of symptoms that would be expected in the absence oftreatment. Beneficial or desired clinical results include, but are notlimited to, alleviation of one or more symptom(s), diminishment ofextent of the deficit, stabilized (i.e., not worsening) state of health,delay or slowing of the disease progression, and amelioration orpalliation of symptoms. Treatment can also include the subject survivingbeyond when mortality would be expected statistically.

As used herein, the term “administering,” refers to the placement of apluripotent cell produced according to the methods described hereinand/or the at least partially differentiated progeny of such apluripotent cell into a subject by a method or route which results in atleast partial localization of the cells at a desired site. Apharmaceutical composition comprising a pluripotent cell producedaccording to the methods described herein and/or the at least partiallydifferentiated progeny of such a pluripotent cell can be administered byany appropriate route which results in an effective treatment in thesubject.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates, for example, include chimpanzees, cynomologousmonkeys, spider monkeys, and macaques, e.g., Rhesus monkeys. Rodentsinclude mice, rats, woodchucks, ferrets, rabbits and hamsters. Domesticand game animals include cows, horses, pigs, deer, bison, buffalo,feline species, e.g., domestic cat, canine species, e.g., dog, fox,wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout,catfish and salmon. Patient or subject includes any subset of theforegoing, e.g., all of the above. In certain embodiments, the subjectis a mammal, e.g., a primate, e.g., a human.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of adisease associated with a deficiency, malfunction, and/or failure of agiven cell or tissue or a deficiency, malfunction, or failure of a stemcell compartment. In addition, the methods described herein can be usedto treat domesticated animals and/or pets. A subject can be male orfemale. A subject can be one who has been previously diagnosed with oridentified as suffering from or having a deficiency, malfunction, and/orfailure of a cell type, tissue, or stem cell compartment or one or morediseases or conditions associated with such a condition, and optionally,but need not have already undergone treatment for such a condition. Asubject can also be one who has been diagnosed with or identified assuffering from a condition including a deficiency, malfunction, and/orfailure of a cell type or tissue or of a stem cell compartment, but whoshows improvements in known risk factors as a result of receiving one ormore treatments for such a condition. Alternatively, a subject can alsobe one who has not been previously diagnosed as having such a condition.For example, a subject can be one who exhibits one or more risk factorsfor such a condition or a subject who does not exhibit risk factors forsuch conditions.

As used herein, the term “select”, when used in reference to a cell orpopulation of cells, refers to choosing, separating, segregating, and/orselectively propagating one or more cells having a desiredcharacteristic. The term “select” as used herein does not necessarilyimply that cells without the desired characteristic are unable topropagate in the provided conditions.

As used herein, “maintain” refers to continuing the viability of a cellor population of cells. A maintained population will have a number ofmetabolically active cells. The number of these cells can be roughlystable over a period of at least one day or can grow.

As used herein, a “detectable level” refers to a level of a substance oractivity in a sample that allows the amount of the substance or activityto be distinguished from a reference level, e.g. the level of substanceor activity in a cell that has not been exposed to a stress. In someembodiments, a detectable level can be a level at least 10% greater thana reference level, e.g. 10% greater, 20% greater, 50% greater, 100%greater, 200% greater, or 300% or greater.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation (2SD) difference above or below a reference, e.g. a concentration orabundance of a marker, e.g. a stem cell marker or differentiationmarker. The term refers to statistical evidence that there is adifference. It is defined as the probability of making a decision toreject the null hypothesis when the null hypothesis is actually true.The decision is often made using the p-value.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

Other terms are defined herein within the description of the variousaspects of the technology described herein.

The aspects of the technology described herein relate to methods ofgenerating a pluripotent cell from a cell as well as uses and methods ofusing those pluripotent cells. In contrast with existing methods ofgenerating pluripotent cells (i.e. induced pluripotent stem cells or iPScells) which rely upon increasing the expression of reprogrammingfactors, for example, by introducing nucleic acid constructs encodingone or more reprogramming factors (e.g. Oct4), the methods describedherein subject the cells to a stress but do not require introduction offoreign reprogramming actors.

In some embodiments, the stress reduces the volume of the cell'scytoplasm and/or the number of the cell's mitochondria. The reduction ofthe volume of the cell's cytoplasm or the number of the cell'smitochondria induces a stress response during which the cell acquires atleast pluripotent capabilities. In one aspect, described herein is amethod to generate a pluripotent cell, comprising removing at leastabout 40% of the cytoplasm from a cell, and selecting cells exhibitingpluripotency, wherein the cell is not present in a tissue. In oneaspect, the invention as described herein relates to a method togenerate a pluripotent cell, comprising removing at least about 40% ofthe mitochondria from a cell, and selecting cells exhibitingpluripotency, wherein the cell is not present in a tissue.

The cells used in the methods, assays, and compositions described hereincan be any type of cell, e.g. an adult cell, an embryonic cell, adifferentiated cell, a stem cell, a progenitor cell, and/or a somaticcell. A cell can be described by combinations of the terms describedabove, e.g. a cell can be an embryonic stem cell or a differentiatedsomatic cell. The cell used in the methods, assays, and compositionsdescribed herein can be obtained from a subject. In some embodiments,the cell is a mammalian cell. In some embodiments, the cell is a humancell. In some embodiments, the cell is an adult cell. In someembodiments, the cell is a neonatal cell. In some embodiments, the cellis a fetal cell. In some embodiments, the cell is an amniotic cell. Insome embodiments, the cell is a cord blood cell.

“Adult” refers to tissues and cells derived from or within an animalsubject at any time after birth. “Embryonic” refers to tissues and cellsderived from or within an animal subject at any time prior to birth.

As used herein, the term “somatic cell” refers to any cell other than agerm cell, a cell present in or obtained from a pre-implantation embryo,or a cell resulting from proliferation of such a cell in vitro. Statedanother way, a somatic cell refers to any cells forming the body of anorganism, as opposed to germline cells. In mammals, germline cells (alsoknown as “gametes”) are the spermatozoa and ova which fuse duringfertilization to produce a cell called a zygote, from which the entiremammalian embryo develops. Every other cell type in the mammalianbody—apart from the sperm and ova, the cells from which they are made(gametocytes) and undifferentiated stem cells—is a somatic cell:internal organs, skin, bones, blood, and connective tissue are all madeup of somatic cells. In some embodiments the somatic cell is a“non-embryonic somatic cell,” by which is meant a somatic cell that isnot present in or obtained from an embryo and does not result fromproliferation of such a cell in vitro. In some embodiments the somaticcell is an “adult somatic cell,” by which is meant a cell that ispresent in or obtained from an organism other than an embryo or a fetusor results from proliferation of such a cell in vitro. It is noted thatadult and neonatal or embryonic cells can be distinguished by structuraldifferences, e.g. epigenetic organization such as methylation patterns.In some embodiments, the somatic cell is a mammalian somatic cell. Insome embodiments, the somatic cell is a human somatic cell. In someembodiments, the somatic cell is an adult somatic cell. In someembodiments, the somatic cell is a neonatal somatic cell.

As used herein, a “differentiated cell” refers to a cell that is morespecialized in its fate or function than at a previous point in itsdevelopment, and includes both cells that are terminally differentiatedand cells that, although not terminally differentiated, are morespecialized than at a previous point in their development. Thedevelopment of a cell from an uncommitted cell (for example, a stemcell), to a cell with an increasing degree of commitment to a particulardifferentiated cell type, and finally to a terminally differentiatedcell is known as progressive differentiation or progressive commitment.In the context of cell ontogeny, the adjective “differentiated”, or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellit is being compared with. Thus, stem cells can differentiate tolineage-restricted precursor cells (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as an cardiomyocyte precursor), and thento an end-stage differentiated cell, which plays a characteristic rolein a certain tissue type, and may or may not retain the capacity toproliferate further.

As used herein, the term “stem cell” refers to a cell in anundifferentiated or partially differentiated state that has the propertyof self-renewal and has the developmental potential to naturallydifferentiate into a more differentiated cell type, without a specificimplied meaning regarding developmental potential (i.e., totipotent,pluripotent, multipotent, etc.). By self-renewal is meant that a stemcell is capable of proliferation and giving rise to more such stemcells, while maintaining its developmental potential. Accordingly, theterm “stem cell” refers to any subset of cells that have thedevelopmental potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retain the capacity, under certain circumstances, to proliferatewithout substantially differentiating. The term “somatic stem cell” isused herein to refer to any stem cell derived from non-embryonic tissue,including fetal, juvenile, and adult tissue. Natural somatic stem cellshave been isolated from a wide variety of adult tissues including blood,bone marrow, brain, olfactory epithelium, skin, pancreas, skeletalmuscle, and cardiac muscle. Exemplary naturally occurring somatic stemcells include, but are not limited to, mesenchymal stem cells andhematopoietic stem cells. In some embodiments, the stem or progenitorcells can be embryonic stem cells. As used herein, “embryonic stemcells” refers to stem cells derived from tissue formed afterfertilization but before the end of gestation, including pre-embryonictissue (such as, for example, a blastocyst), embryonic tissue, or fetaltissue taken any time during gestation, typically but not necessarilybefore approximately 10-12 weeks gestation. Most frequently, embryonicstem cells are totipotent cells derived from the early embryo orblastocyst. Embryonic stem cells can be obtained directly from suitabletissue, including, but not limited to human tissue, or from establishedembryonic cell lines. In one embodiment, embryonic stem cells areobtained as described by Thomson et al. (U.S. Pat. Nos. 5,843,780 and6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff,1998; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995 which are incorporatedby reference herein in their entirety).

Exemplary stem cells include embryonic stem cells, adult stem cells,pluripotent stem cells, neural stem cells, liver stem cells, muscle stemcells, muscle precursor stem cells, endothelial progenitor cells, bonemarrow stem cells, chondrogenic stem cells, lymphoid stem cells,mesenchymal stem cells, hematopoietic stem cells, central nervous systemstem cells, peripheral nervous system stem cells, and the like.Descriptions of stem cells, including method for isolating and culturingthem, maybe found in, among other places, Embryonic Stem Cells, Methodsand Protocols, Turksen, ed., Humana Press, 2002; Weisman et al., Annu.Rev. Cell. Dev. Biol. 17:387 403; Pittinger et al., Science, 284:143 47,1999; Animal Cell Culture, Masters, ed., Oxford University Press, 2000;Jackson et al., PNAS 96(25):14482 86, 1999; Zuk et al., TissueEngineering, 7:211 228, 2001 (“Zuk et al.”); Atala et al., particularlyChapters 33 41; and U.S. Pat. Nos. 5,559,022, 5,672,346 and 5,827,735.Descriptions of stromal cells, including methods for isolating them, maybe found in, among other places, Prockop, Science, 276:71 74, 1997;Theise et al., Hepatology, 31:235 40, 2000; Current Protocols in CellBiology, Bonifacino et al., eds., John Wiley& Sons, 2000 (includingupdates through March, 2002); and U.S. Pat. No. 4,963,489.

As used herein, “progenitor cells” refers to cells in anundifferentiated or partially differentiated state and that have thedevelopmental potential to differentiate into at least one moredifferentiated phenotype, without a specific implied meaning regardingdevelopmental potential (i.e., totipotent, pluripotent, multipotent,etc.) and that does not have the property of self-renewal. Accordingly,the term “progenitor cell” refers to any subset of cells that have thedevelopmental potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype. In someembodiments, the stem or progenitor cells are pluripotent stem cells. Insome embodiments, the stem or progenitor cells are totipotent stemcells.

The term “totipotent” refers to a stem cell that can give rise to anytissue or cell type in the body. “Pluripotent” stem cells can give riseto any type of cell in the body except germ line cells. Stem cells thatcan give rise to a smaller or limited number of different cell types aregenerally termed “multipotent.” Thus, totipotent cells differentiateinto pluripotent cells that can give rise to most, but not all, of thetissues necessary for fetal development. Pluripotent cells undergofurther differentiation into multipotent cells that are committed togive rise to cells that have a particular function. For example,multipotent hematopoietic stem cells give rise to the red blood cells,white blood cells and platelets in the blood.

The term “pluripotent” as used herein refers to a cell with thecapacity, under different conditions, to differentiate to cell typescharacteristic of all three germ cell layers (i.e., endoderm (e.g., guttissue), mesoderm (e.g., blood, muscle, and vessels), and ectoderm(e.g., skin and nerve)). Pluripotent cells are characterized primarilyby their ability to differentiate to all three germ layers, using, forexample, a nude mouse teratoma formation assay. Pluripotency is alsoevidenced by the expression of embryonic stem (ES) cell markers,although the preferred test for pluripotency is the demonstration of thecapacity to differentiate into cells of each of the three germ layers.

The “ACC” and “STAP” cells described in the Examples herein, arenon-limiting examples of pluripotent cells. The “STAP stem cells” arenon-limiting examples of pluripotent stem cells. The term pluripotentcell and the term pluripotent stem cell may be used hereininterchangeably because both cells can be used suitably for the purposeof the present invention.

The term “pluripotency” or a “pluripotent state” as used herein refersto a cell with the ability to differentiate into all three embryonicgerm layers: endoderm (gut tissue), mesoderm (including blood, muscle,and vessels), and ectoderm (such as skin and nerve).

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and non-limiting examples of multipotent cells can includeadult stem cells, such as for example, hematopoietic stem cells andneural stem cells. Multipotent means a stem cell may form many types ofcells in a given lineage, but not cells of other lineages. For example,a multipotent blood stem cell can form the many different types of bloodcells (red, white, platelets, etc. . . . ), but it cannot form neurons.The term “multipotency” refers to a cell with the degree ofdevelopmental versatility that is less than totipotent and pluripotent.

The term “totipotency” refers to a cell with the degree ofdifferentiation describing a capacity to make all of the cells in theadult body as well as the extra-embryonic tissues including theplacenta. The fertilized egg (zygote) is totipotent as are the earlycleaved cells (blastomeres)

The cell used in the methods described herein can be a cell which is notpresent in a tissue. As used herein, a “tissue” refers to an organizedbiomaterial (e.g. a group, layer, or aggregation) of similarlyspecialized cells united in the performance of at least one particularfunction. When cells are removed from an organized superstructure, orotherwise separated from an organized superstructure which exists invivo, they are no longer present in a tissue. For example, when a bloodsample is separated into two or more non-identical fractions, or aspleen is minced and mechanically-dissociated with Pasteur pipettes, thecells are no longer present in a tissue. In some embodiments, cellswhich are not present in a tissue are isolated cells. The term“isolated” as used herein in reference to cells refers to a cell that ismechanically or physically separated from another group of cells withwhich they are normally associated in vivo. Methods for isolating one ormore cells from another group of cells are well known in the art. See,e.g., Culture of Animal Cells: a manual of basic techniques (3rdedition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: alaboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A.Leinwand (eds.), Cold Spring Harbor Laboratory Press; Animal Cells:culture and media, 1994, D. C. Darling, S. J. Morgan, John Wiley andSons, Ltd. Optionally the isolated cell has been cultured in vitro,e.g., in the presence of other cells.

In some embodiments, a cell, while not present in a tissue, is presentin a population of cells. In some embodiments, the population of cellsis a population of cells. As used herein, a “population of cells” refersto a group of at least 2 cells, e.g. 2 cells, 3 cells, 4 cells, 10cells, 100 cells, 1000 cells, 10,000 cells, 100,000 cells or any valuein between, or more cells. Optionally, a population of cells can becells which have a common origin, e.g. they can be descended from thesame parental cell, they can be clonal, they can be isolated from ordescended from cells isolated from the same tissue, or they can beisolated from or descended from cells isolated from the same tissuesample. A population of cells can comprise 1 or more cell types, e.g. 1cell type, 2 cell types, 3 cell types, 4 cell types or more cell types.A population of cells can be heterogeneous or homogeneous. A populationof cells can be substantially homogeneous if it comprises at least 90%of the same cell type, e.g. 90%, 92%, 95%, 98%, 99%, or more of thecells in the population are of the same cell type. A population of cellscan be heterogeneous if less than 90% of the cells present in thepopulation are of the same cell type.

In some embodiments, the methods described herein can relate to making anon-pluripotent cell (e.g. a differentiated cell) assume a pluripotentphenotype. In some embodiments, generating a pluripotent cell caninclude generating a cell with a more pluripotent phenotype, i.e.causing a cell to assume a phenotype which has broader differentiationpotential. By way of non-limiting example, very small embryonic-likecells (VSEL) cells can be unipotent instead of pluripotent, and/or belimited in their ability to differentiate into certain differentiatedcell types (possibly due the epigenetic state of VSELs more closelyresembling differentiated cells than embryonic stem cells). Inaccordance with the methods described herein, a unipotent cell and/orcell with limited differentiation ability can be caused to assume a morepluripotent phenotype. A more pluripotent phenotype can be a phenotypethat is able to differentiate into a greater number of differentiatedcell types e.g. of two unipotent cells, the one that can differentiateinto a greater number of differentiated cell types of that lineage ismore pluripotent and/or a pluripotent cell is more pluripotent than aunipotent cell.

The methods of generating a pluripotent cell (or more pluripotent cell)described herein can comprise, for example, removing part of thecytoplasm from a cell and/or removing mitochondria from a cell. In someembodiments, the removal of part of the cytoplasm or mitochondria from acell removes partial epigenetic control of the cell. In someembodiments, at least about 40% of the cytoplasm is removed, e.g. atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90% or more of the cytoplasm ofa cell is removed. In some embodiments, between 60% and 80% of thecytoplasm of a cell is removed. In some embodiments, at least about 40%of the mitochondria are removed, e.g. at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90% or more of the mitochondria of a cell are removed. Insome embodiments, between 50% and 90% of the mitochondria of a cell areremoved.

The method of subjecting the cell to stress and/or removing part of thecytoplasm or mitochondria from a cell can be any environmental stimulusthat will cause pores and/or ruptures in the membrane of a cell belowthe threshold of lethality. The stress may comprise unphysiologicalstress in tissue or cell culture. Non-limiting examples of suitableenvironmental stimuli include trauma, mechanical stimuli, chemicalexposure, ultrasonic stimulation, oxygen-deprivation,nutrient-deprivation, radiation, exposure to extreme temperatures,dissociation, trituration, physical stress, hyper osmosis, hypo osmosis,membrane damage, toxin, extreme ion concentration, active oxygen, IJVexposure, strong visible light, deprivation of essential nutrition, orunphysiologically acidic environment. In some embodiments, oneenvironmental stimulus can be applied to a cell. In some embodiments,multiple environmental stimuli can be applied to a cell, e.g. 2 stimuli,3 stimuli, 4 stimuli or more stimuli can be applied. Multipleenvironmental stimuli can be applied concurrently or separately.

In some embodiments, the stress can be a stress that will cause membranedisruption in at least 10% of the cells exposed to the stress. As usedherein, “membrane disruption” refers to compromising, rupturing, ordisrupting a membrane such that pores or gaps form, sufficient toreleased a detectable amount of organelles and/or cellular material,including but not limited to mitochondria and DNA into the extracellularenvironment. Methods of detecting the release of cellular material, e.g.mitochondria are known in the art and described elsewhere herein. Thereleased cellular material can be free or encapsulated or surrounded bymembranes.

The stress can cause membrane disruption in at least 10% of the cellsexposed to the stress, e.g. 10% or more, 20% or more, 30% or more, 40%or more 50% or more, 60% or more, 70% or more, 80% or more, or 90% ormore. In some embodiments, the cells exposed to the stress can be cellsof the same type and characteristics as the cells to be made morepluripotent as described herein, e.g. the stress suitable for one typeof cell may not be suitable for another type of cell.

The length of time for which the cells are exposed to stress can varydepending upon the stimulus being used. For example, when using lownutrition conditions to stress cells according to the methods describedherein, the cells can be cultured under low nutrition conditions for 1week or more, e.g. 1 week, 2 weeks, or 3 weeks or longer. In someembodiments, the cells are cultured under low nutrition conditions forabout 3 weeks. In another non-limiting example, cells exposed to low pHor hypoxic conditions according to the methods described herein can beexposed for minutes or long, e.g. including for several hours, e.g. forat least 2 minutes, for at least 5 minutes, for at least 20 minutes, forat least 1 hour, for at least 2 hours, for at least 6 hours or longer.

Mechanical stimuli that induce the generation of pluripotent cells caninclude any form of contact of a substance or surface with the cellmembrane which will mechanically disrupt the integrity of the membrane.Mechanical stimulus can comprise exposing the cell to shear stressand/or high pressure. An exemplary form of mechanical stimulus istrituration. Trituration is a process of grinding and/or abrading thesurface of a particle via friction. A non-limiting example of a processfor trituration of a cell is to cause the cell to pass through a devicewherein the device has an aperture smaller than the size of the cell.For example, a cell can be caused, by vacuum pressure and/or the flow ofa fluid, to pass through a pipette in which at least part of theinterior space of the pipette has a diameter smaller than the diameterof the cell. In some embodiments, the cell is passed through at leastone device with a smaller aperture than the size of the cell. In someembodiments, the cell is passed through several devices havingprogressively smaller apertures. In some embodiments, cells can betriturated for 5 or more minutes, e.g. 5 minutes, 10 minutes, 20minutes, 30 minutes, or 60 minutes. In some embodiments, the cells canbe triturated by passing them through a Pasteur pipette with an internaldiameter of 50 μm. In some embodiments, the cells can be triturated bypassing them through a Pasteur pipette with an internal diameter of 50μm for 20 minutes.

Other methods of applying stress necessary to induce cells to generatepluripotent cells include, for example, exposure to certain chemicals,or physico-chemical conditions (e.g. high or low pH, osmotic shock,temperature extremes, oxygen deprivation, etc). Treatments of this kindand others that induce the generation of pluripotent cells are discussedfurther below. Chemical exposure can include, for example, anycombination of pH, osmotic pressure, and/or pore-forming compounds thatdisrupt or compromise the integrity of the cell membrane. By way ofnon-limiting example, the cells can be exposed to unphysiolosicallyacidic environment or low pH, streptolysin O, or distilled water (i.e.osmotic shock).

Low pH can include a pH lower than 6.8, e.g. 6.7, 6.5, 6.3, 6.0, 5.8,5.4, 5.0, 4.5, 4.0, or lower. In some embodiments, the low pH is fromabout 3.0 to about 6.0. In some embodiments, the low pH is from about4.5 to about 6.0. In some embodiments, the low pH is from 5.4 to 5.8. Insome embodiments, the low pH is from 5.4 to 5.6. In some embodiments,the low pH is about 5.6. In some embodiments, the low pH is about 5.7.In some embodiments, the low pH is about 5.5. In some embodiments, thecells can be exposed to low pH conditions for up to several days, e.g.for 6 days or less, for 4 days or less, for 3 days or less, for 2 daysor less, for 1 day or less, for 12 hours or less, for 6 hours or less,for 3 hours or less, for 2 hours or less, for 1 hour or less, for 30minutes or less, for 20 minutes or less, or less than 10 minutes. Insome embodiments, the cells can be exposed to a pH from 5.4 to 5.6 for 3days or less. In some embodiments, the cells can be exposed to a pH offrom about 5.6 to 6.8 for 3 days or less. In some embodiments, the cellscan be exposed of a pH of from about 5.6 to 6.8 for 1 hour or less. Insome embodiments, the cells can be exposed of a pH of from about 5.6 to6.8 for about 30 minutes. In some embodiments, the cells can be exposedof a pH of from about 5.6 to 6.8 for about 20 minutes. In someembodiments, the cells can be exposed to a pH of from about 5.6 to 5.8for 3 days or less. In some embodiments, the cells can be exposed of apH of from about 5.6 to 5.8 for 1 hour or less. In some embodiments, thecells can be exposed of a pH of from about 5.6 to 5.8 for about 30minutes. In some embodiments, the cells can be exposed of a pH of fromabout 5.6 to 5.8 for about 20 minutes.

In some embodiments, cells can be exposed to ATP to induce thegeneration of pluripotent cells. In some embodiments, cells can beexposed to ATP at concentrations from about 20 μM to about 200 mM. Insome embodiments, cells can be exposed to ATP at concentrations fromabout 200 μM to about 20 mM. In some embodiments, cells can be exposedto ATP at concentrations of about 2.4 mM. In some embodiments, cell canbe exposed to ATP diluted in HBSS. In some embodiments, cells can beexposed to ATP for 1 minute or longer, e.g. at least 1 minute, at least2 minutes, at least 5 minutes, at least 15 minutes, at least 30 minutes,at least 45 minutes, at least 1 hour or longer. In some embodiments, thecells can be exposed to ATP for from about 5 minutes to about 30minutes. In some embodiments, the cells can be exposed to ATP for about15 minutes. In some embodiments, the cells can be exposed to about 2.4mM ATP for about 15 minutes.

In some embodiments, cells can be exposed to CaCl₂ to induce thegeneration of pluripotent cells. In some embodiments, cells can beexposed to CaCl₂ at concentrations from about 20 μM to about 200 mM. Insome embodiments, cells can be exposed to CaCl₂ at concentrations fromabout 200 μM to about 20 mM. In some embodiments, cells can be exposedto CaCl₂ at concentrations of about 2 mM. In some embodiments, cells canbe exposed to CaCl₂ diluted in HBSS. In some embodiments, cells can beexposed to CaCl₂ for 1 day or longer, e.g. at least 1 day, at least 2days, at least 1 week, at least 2 weeks, at least 3 weeks or longer. Insome embodiments, the cells can be exposed to CaCl₂ for from about 1week to 3 weeks. In some embodiments, the cells can be exposed to CaCl₂for about 2 weeks. In some embodiments, the cells can be exposed toabout 2 mM CaCl₂ for about 2 weeks. In some embodiments, the cells canbe exposed to about 2 mM CaCl₂ for about 1 week.

Examples of pore-forming compounds include streptolysin O (SLO),saponin, digitonin, filipin, Ae I, cytolysin of sea anemone, aerolysin,amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar,brevinin-1E, brevinin-2E, barbatolysin, cytolysin of Enterococcusfaecalis, delta hemolysin, diphtheria toxin, El Tor cytolysin of Vibriocholerae, equinatoxin, enterotoxin of Aeromonas hydrophila, esculentin,granulysin, haemolysin of Vibrio parahaemolyticus, intermedilysin ofStreptococcus intermedins, the lentivirus lytic peptide, leukotoxin ofActinobacillus actinomycetemcomitans, magainin, melittin,membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin,nexkyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphinfragment 3, neokyotorphin fragment 4, NKlysin, paradaxin, alphacytolysin of Staphylococcus aureus, alpha cytolysin of Clostridiumsepticum, Bacillus thuringiensin toxin, colicin, complement, defensin,histolysis, listeriolysin, magainin, melittin, pneumolysin, yeast killertoxin, valinomycin, Peterson's crown ethers, perforin, perfringolysin O,theta-toxin of Clostridium perfringens, phallolysin, phallotoxin, andother molecules, such as those described in Regen et al. Biochem BiophysRes Commun 1989 159:566-571; which is incorporated herein by referencein its entirety. Methods of purifying or synthesizing pore-formingcompounds are well known to one of ordinary skill in the art. Further,pore-forming compounds are commercially available, e.g. streptolysin O(Cat No. S5265; Sigma-Aldrich; St. Louis, Mo.). Byway of non-limitingexample, cells can be exposed to SLO for about 5 minutes or more, e.g.,at least 5 minutes, at least 10 minutes, at least 20 minutes, at least30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, atleast 3 hours, or longer. In some embodiments. cells are exposed to SLOfor from about 30 minutes to 2 hours. In some embodiments, cells areexposed to SLO for about 50 minutes. By way of non-limiting example,cells can be exposed to SLO at concentrations of from about 10 ng/mL to1 mg/mL. In some embodiments, cells can be exposed to SLO atconcentrations of from about 1 μg/mL to 100 μg/mL. In some embodiments,cells can be exposed to SLO at about 10 μg/mL. In some embodiments,cells can be exposed to SLO at about 10 μg/mL for about 50 minutes.

Oxygen-deprivation conditions that induce the generation of pluripotentcells can include culturing cells under reduced oxygen conditions, e.g.culturing cells in 10% oxygen or less. In some embodiments, the cellsare cultured under 5% oxygen or less. The length of culturing underreduced oxygen conditions can be 1 hour or longer, e.g. 1 hour, 12hours, 1 day, 2 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months orlonger. In some embodiments, the cells can be cultured under reducedoxygen conditions for from 1 week to 1 month. In some embodiments, thecells can be cultured under reduced oxygen conditions for about 3 weeks.

Nutrient-deprivation conditions that induce the generation ofpluripotent cells can include the lack of any factor or nutrient that isbeneficial to cell growth. In some embodiments, nutrient-deprivationconditions comprise culturing the cells in basal culture medium, e.g.F12 or DMEM without further supplements such as FBS or growth factors.The length of culturing in nutrient-deprivation conditions can be 1 houror longer, e.g. 1 hour, 12 hours, 1 day, 2 days, 1 week, 2 weeks, 3weeks, 1 month, 2 months or longer. In some embodiments, the cells canbe cultured under nutrient-deprivation conditions for from 1 week to 1month. In some embodiments, the cells can be cultured undernutrient-deprivation conditions for about 2 weeks. In some embodiments,the cells can be cultured under nutrient-deprivation conditions forabout 3 weeks. In some embodiments, nutrient-deprivation conditions caninclude conditions with no growth factors or conditions with less than50% of a standard concentration of one or more growth factors for agiven cell type.

Exposure to extreme temperatures that induces the generation ofpluripotent cells can include exposure to either low temperatures orhigh temperatures. For a mammalian cell, an extreme low temperature canbe a temperature below 35° C., e.g. 34° C., 33° C., 32° C., 31° C., orlower. In some embodiments, an extreme low temperature can be atemperature below freezing. Freezing of cells can cause membraneperforations by ice crystals and provides an avenue for reducingcytoplasm. For a mammalian cell, an extreme high temperature can be atemperature above 42° C., e.g. 43° C., 44° C., 45° C., 46° C. or higher.In some embodiments, the extreme high temperature can be a temperatureof about 85° C. or higher. The length of culturing under extremetemperatures can be 20 minutes or longer, e.g. 20 minutes, 30 minutes, 1hour, 12 hours, 1 day, 2 days, 1 week, 2 weeks, 3 weeks, 1 month, 2months or longer. Clearly, the higher the temperature, the shorter theexposure that will generally be tolerated to permit the generation ofpluripotent cells.

Further examples of stresses that can be used in the methods describedherein include, but are not limited to, ultrasonic stimulation andradiation treatment.

In some embodiments, after being exposed to a stress, the cells can becultured prior to selection according to the methods described belowherein. The cells can be cultured for at least 1 hour prior toselection, e.g. the stressful stimulus is removed and the cells arecultured for at least 1 hour, at least 2 hours, at least 6 hours, atleast 12 hours, at least 1 day, at least 2 days, at least 7 days orlonger prior to selecting as described herein. By way of non-limitingexample, cells can be exposed to SLO for about 50 minutes and thencultured in culture medium without SLO for about 7 days prior toselection. In some embodiments, the culture medium used to culture thecells prior to selection does not contain differentiation factors orpromote differentiation. In some embodiments, the culture medium is onesuitable for the culture of stem cells and/or pluripotent cells.Examples of such media are described below herein.

In some embodiments, the amount of cytoplasm in a cell is reduced. Thereduction of cytoplasm in a cell can be determined by monitoring thesize of the cell. Methods of determining cell size are well known to oneof ordinary skill in the art and include, by way of non-limitingexample, cytofluorimetric analysis. In brief, single cells are stainedwith propidium iodide filtered and measured, for example, on a DAKOGALAXY™ (DAKO) analyzer using FLOMAX™ software. Cytofluorimetricanalysis can then be performed to establish cell size. Microbeads ofpredefined sizes are re-suspended in isotonic phosphate saline (pH 7.2)and used as a standard for which to compare size of cells contained inspheres using cytofluorimetric analysis. Both cells and beads areanalyzed using the same instrument setting (forward scatter,representing cell and bead size, and side scatter, representing cellulargranularity). Cell size can be calculated on a curve employing bead sizeon the x-axis and forward scatter values on the y-axis.

In some embodiments, the amount of mitochondria in a cell is reduced.Methods of determining the number of mitochondria in a cell are wellknown to one of ordinary skill in the art and include staining with amitochondria-specific dye and counting the number of mitochondriavisible per cell when viewed under a microscope. Mitochondria-specificdyes are commercially available, e.g. MITOTRACKER™ (Cat No M7512Invitrogen; Grand Island, N.Y.). In some embodiments, the number ofmitochondria or the intensity of the signal from mitochondria-specificdyes can be decreased by at least 40% following treatment with themethods described above herein. In some embodiments, cells are selectedin which the number of mitochondria or the intensity of the signal frommitochondria-specific dyes decreased by at least 40% following treatmentwith the methods described above herein.

The amount of mitochondria and/or membrane disruption can also bedetected by measuring redox activity in the extracellular environment.As mitochondria are released into the extracellular environment by thestress described herein, the level of ROS in the extracellularenvironment can increase and can be used to measure the effectiveness ofa given stress.

In some embodiments of any of the aspects described herein, the cell canbe subjected to a stress while in the presence of LIF (leukemiainhibitory factor).

In some aspects, after removing a portion of the cytoplasm and/ormitochondria of a cell, the method further comprises selecting cellsexhibiting pluripotency. Pluripotent cells can be selected by selectingcells which display markers, phenotypes, or functions of pluripotentcells. Selecting cells can comprise isolating and propagating cellsdisplaying the desired characteristics or culturing a population ofcells with unknown characteristics under conditions such that cells withthe desired characteristic(s) will survive and/or propagate at a higherrate than those cells not having the desired characteristic(s).Non-limiting examples of markers and characteristics of pluripotentcells are described herein below. In some embodiments, selecting thecells for pluripotency comprises, at least in part, selecting cellswhich express Oct4. In some embodiments, selecting the cells forpluripotency comprises, at least in part, selecting cells which expressNanog. In some embodiments, selecting the cells for pluripotencycomprises, at least in part, selecting cells which express Oct4, Nanog,E-cadherin, and/or SSEA. In some embodiments, pluripotent cells can beselected by selecting cells expressing SSEA-1 and E-cadherin usingantibodies specific for those markers and FACS. In some embodimentscells can be selected on the basis of size using FACS or other cellsorting devices as known in the art and/or described herein. Cells canalso be selected by their inability to adhere to culture dishes.

Cells can also be selected on the basis of smaller size after beingsubjected to stress. That is, stressed cells that progress topluripotency are smaller than their non-pluripotent somatic precursors.In some embodiments, cells with a diameter of less than 8 μm areselected, e.g. cells with a diameter of 8 μm or less, 7 μm or less, 6 μmor less, 5 μm or less, or smaller. Cells can be selected on the basis ofsize after being cultured for a brief period (e.g. several minutes toseveral days) or after being allowed to rest following the stresstreatment. In some embodiments, the cells can be selected on the basisof size immediately following the stress treatment. Cells can beselected on the basis of size by any method known in the art, e.g. theuse of a filter or by FACS.

In some embodiments of the methods described herein, a pluripotent cellgenerated according to the methods described herein can be cultured topermit propagation of that pluripotent cell (i.e. propagation of a stemcell). In some embodiments, a pluripotent cell generated according tothe methods described herein can be maintained in vitro. In one aspect,the technology described herein relates to a composition comprising apluripotent cell and/or the at least partially differentiated progenythereof. In some embodiments, the pluripotent cell and/or the at leastpartially differentiated progeny thereof can be maintained in vitro,e.g. as a cell line. Cell lines can be used to screen for and/or testcandidate agents, e.g. therapeutic agents for a given disease and/oragents that modulate stem cells, as described below herein. In someembodiments, the pluripotent cell and/or the at least partiallydifferentiated progeny thereof can be derived from a cell obtained froma subject with a disease, e.g. a disease associated with the failure ofa naturally occurring cell or tissue type or a naturally occurringpluripotent and/or multipotent cell (as described herein below), and/ora disease involving cells which have genetic mutations, e.g. cancer. Thecompositions described herein, can be used, e.g. in disease modeling,drug discovery, diagnostics, and individualized therapy.

Conditions suitable for the propagation and or maintaining of stemand/or pluripotent cells are known in the art. Propagation of stem cellspermits expansion of cell numbers without substantially inducing orpermitting differentiation By way of non-limiting example, conditionssuitable for propagation of pluripotent cells include plating cells at1×10⁶ cells/cm² in F12/DMEM (1:1, v/v) supplemented with 2% B27, 20ng/mL basic fibroblast growth factor, and 10 ng/mL epidermal growthfactor. About 50% of the medium can be replaced every 2-3 days for theduration of the culture. In some embodiments, the conditions suitablefor the propagation of stem and/or pluripotent cells comprise culturingthe cells in B27-LIF (i.e. serum-free medium containing LIF (1×10³units/mL, Chemicom; Cat No: ESG1107 EMD Millipore, Billerica, Mass.) andB27 supplement (Cat No: 0080085-SA; Invitrogen; Grand island, NY) asdescribed in Hitoshi, S. et al. Genes & development 2004 18, 1806-1811;which is incorporated by reference herein in its entirety. Other media,suitable for culturing the cells described herein are described in theExamples herein, e.g. ES establishment culture medium, 2i, 3i and ACTH,ES culture condition, ES-LIF, embryonic neural stem cell culturecondition, and EpiSCs culture condition. In some embodiments, conditionsfor the propagation or maintenance of pluripotent cells can includeculture the cells in the presence of LIF (leukemia inhibitory factor).

During propagation, the pluripotent cell generated according to themethods described herein will continue to express the same pluripotentstem cell marker(s). Non-limiting examples of pluripotent stem cellmarkers include SSEA-1, SSEA-2, SSEA-3, SSEA-4 (collectively referred toherein as SSEA), AP, E-cadherin antigen, Oct4, Nanog, Ecat1, Rex1,Zfp296, GDF3, Dppa3, Dppa4, Dppa5, Sox2, Esrrb, Dnmt3b, Dnmt31, Utf1,Tcl1, Bat1, Fgf4, Neo, Cripto, Cdx2, and Slc2a3. Methods of determiningif a cell is expressing a pluripotent stem cell marker are well known toone of ordinary skill in the art and include, for example, RT-PCR, theuse of reporter gene constructs (e.g. expression of the Oct4-GFPconstruct described herein coupled with FACS or fluorescencemicroscopy), and FACS or fluorescence microscopy using antibodiesspecific for cell surface markers of interest.

Pluripotent cell markers also include elongated telomeres, as comparedto cells. Telomere length can be determined, for example, by isolatinggenomic DNA, digesting the gDNA with restriction enzymes such as Hinf1and Rsa1, and detecting telomeres with a telomere length assay reagent.Such reagents are known in the art and are commercially available, e.g.the TELOTAGGG™ TELOMERE LENGTH ASSAY kit (Cat No. 12209136001 Roche;Indianapolis, Ind.).

In some embodiments, a cell treated according to the methods describedherein can be altered to more closely resemble the epigenetic state ofan embryonic stem cell than it did prior to being treated in accordancewith the disclosed methods. The epigenetic state of a cell refers to thechemical marking of the genome, as opposed to changes in the nucleotidesequence of the genome. Epigenetic marks can include DNA methylation(imprints) as well as methylation and acetylation of proteins associatedwith DNA, such as histones. The term ‘DNA methylation’ refers to theaddition of a methyl (CH₃) group to a specific base in the DNA. Inmammals, methylation occurs almost exclusively at the 5 position on acytosine when this is followed by a guanine (CpG). In some embodiments,the epigenetic state can comprise epigenetic methylation patterns, e.g.DNA methylation patterns. Assays for determining the presence andlocation of epigenetic markings are known in the art, and can includebisulfite sequencing, e.g. as described in Example 2 herein. Briefly,DNA is treated with the CpGenome™ DNA Modification Kit (Chemicon,Temecula, Calif.,) and regions of interest (e.g. the Nanog and Oct4genes) are amplified and sequenced.

Some aspects of the technology described herein relate to assays using apluripotent stem cell produced by the methods described herein. Forexample, a pluripotent stem cell produced by the methods describedherein can be used to screen and/or identify agents which modulate theviability, differentiation, or propagation of pluripotent stem cells.Such assays can comprise contacting a pluripotent cell producedaccording to the methods described herein with a candidate agent anddetermining whether the viability, differentiation and/or propagation ofthe pluripotent cell contacted with the candidate agent varies from theviability, differentiation and/or propagation of a pluripotent cell notcontacted with the candidate agent. In some embodiments, an agent canincrease the viability, differentiation, and/or propagation of thepluripotent stem cell. In some embodiments, an agent can decrease theviability, differentiation, and/or propagation of the pluripotent stemcell. In some embodiments, the pluripotent stem cell can be contactedwith multiple candidate agents, e.g. to determine synergistic orantagonistic effects or to screen candidate agents in pools.

A candidate agent is identified as an agent that modulates the viabilityof a pluripotent cell produced if the number of pluripotent cells whichare viable, i.e. alive is higher or lower in the presence of thecandidate agent relative to its absence. Methods of determining theviability of a cell are well known in the art and include, by way ofnon-limiting example determining the number of viable cells at at leasttwo time points, by detecting the strength of a signal from a live cellmarker, or the number or proportion of cells stained by a live cellmarker. Live cell markers are available commercially, e.g. PRESTO BLUE™(Cat No A-13261; Life Technologies; Grand Island, N.Y.). A candidateagent is identified as an agent that modulates the propagation of apluripotent cell produced if the rate of propagation of the pluripotentcell is altered, i.e. the number of progeny cells produced in a giventime is higher or lower in the presence of the candidate agent. Methodsof determining the rate of propagation of a cell are known in the artand include, by way of non-limiting example, determining an increase inlive cell number over time.

A candidate agent is identified as an agent that modulates thedifferentiation of a pluripotent cell if the rate or character of thedifferentiation of the pluripotent cell is higher or lower in thepresence of the candidate agent. Methods of determining the rate orcharacter of differentiation of a cell are known in the art and include,by way of non-limiting example, detecting markers or morphology of aparticular lineage and comparing the number of cells and/or the rate ofappearance of cells with such markers or morphology in the populationcontacted with a candidate agent to a population not contacted with thecandidate agent. Markers and morphological characteristics of variouscell fate lineages and mature cell types are known in the art. By way ofnon-limiting example, mesodermal cells are distinguished frompluripotent cells by the expression of actin, myosin, and desminChondrocytes can be distinguished from their precursor cell types bystaining with safranin-O and or FASTGREEN™ dyes (Fisher; Pittsburgh,Pa.; F99). Osteocytes can be distinguished from their precursor celltypes by staining with Alizarin Red S (Sigma; St. Louis, Mo.: Cat NoA5533).

In some embodiments, a candidate agent can be an potential inhibitor oftumor stem cells, e.g. the methods described herein can be used tocreate pluripotent cells from mature tumor cells, and used to screen foragents which inhibit the creation and/or viability of tumor cells. Themethods described herein can also be used to screen for agents whichkill mature tumor cells but which do not promote the development and/orsurvival of tumor stem cells.

In some embodiments, the pluripotent cells are contacted with one ormore candidate agents and cultured under conditions which promotedifferentiation to a particular cell lineage or mature cell type.Conditions suitable for differentiation are known in the art. By way ofnon-limiting example, conditions suitable for differentiation to themesoderm lineage include DMEM supplemented with 20% fetal calf serum(FCS), with the medium exchanged every 3 days. By way of furthernon-limiting example, conditions suitable for differentiation to theneural lineage include plating cells on ornithin-coated chamber slidesin F12/DMEM (1:1, v/v) supplemented 2% B27, 10% FCS, 10 ng/mL bFGF, and20 ng/m LEGF. The medium can be exchanged every 3 days.

As used herein, a “candidate agent” refers to any entity which isnormally not present or not present at the levels being administered toa cell, tissue or subject. A candidate agent can be selected from agroup comprising: chemicals; small organic or inorganic molecules;nucleic acid sequences; nucleic acid analogues; proteins; peptides;aptamers; peptidomimetic, peptide derivative, peptide analogs,antibodies; intrabodies; biological macromolecules, extracts made frombiological materials such as bacteria, plants, fungi, or animal cells ortissues; naturally occurring or synthetic compositions or functionalfragments thereof. In some embodiments, the candidate agent is anychemical entity or moiety, including without limitation synthetic andnaturally-occurring non-proteinaceous entities. In certain embodimentsthe candidate agent is a small molecule having a chemical moiety. Forexample, chemical moieties include unsubstituted or substituted alkyl,aromatic, or heterocyclyl moieties including macrolides, leptomycins andrelated natural products or analogues thereof. Candidate agents can beknown to have a desired activity and/or property, or can be selectedfrom a library of diverse compounds.

Candidate agents can be screened for their ability to modulate theviability, propagation, and/or differentiation of a pluripotent cell. Inone embodiment, candidate agents are screened using the assays forviability, differentiation, and/or propagation described above and inthe Examples herein.

Generally, compounds can be tested at any concentration that canmodulate cellular function, gene expression or protein activity relativeto a control over an appropriate time period. In some embodiments,compounds are tested at concentrations in the range of about 0.1 nM toabout 1000 mM. In one embodiment, the compound is tested in the range ofabout 0.1 μM to about 20 μM, about 0.1 μM to about 10 μM, or about 0.1μM to about 5 μM.

Depending upon the particular embodiment being practiced, the candidateor test agents can be provided free in solution, or can be attached to acarrier, or a solid support, e.g., beads. A number of suitable solidsupports can be employed for immobilization of the test agents. Examplesof suitable solid supports include agarose, cellulose, dextran(commercially available as, e.g., Sephadex, Sepharose) carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper,nitrocellulose, ion exchange resins, plastic films,polyaminemethylvinylether maleic acid copolymer, glass beads, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc.Additionally, for the methods described herein, test agents can bescreened individually, or in groups or pools. Group screening isparticularly useful where hit rates for effective test agents areexpected to be low, such that one would not expect more than onepositive result for a given group.

Methods for developing small molecule, polymeric and genome basedlibraries are described, for example, in Ding, et al. J Am. Chem. Soc.124: 1594-1596 (2002) and Lynn, et al., J. Am. Chem. Soc. 123: 8155-8156(2001). Commercially available compound libraries can be obtained from,e.g., ArQule (Woburn, Mass.), Invitrogen (Carlsbad, Calif.), RyanScientific (Mt. Pleasant, S.C.), and Enzo Life Sciences (Farmingdale,N.Y.). These libraries can be screened for the ability of members tomodulate the viability, propagation, and/or differentiation ofpluripotent stem cells. The candidate agents can be naturally occurringproteins or their fragments. Such candidate agents can be obtained froma natural source, e.g., a cell or tissue lysate. Libraries ofpolypeptide agents can also be prepared, e.g., from a cDNA librarycommercially available or generated with routine methods. The candidateagents can also be peptides, e.g., peptides of from about 5 to about 30amino acids, with from about 5 to about 20 amino acids being preferredand from about 7 to about 15 being particularly preferred. The peptidescan be digests of naturally occurring proteins, random peptides, or“biased” random peptides. In some methods, the candidate agents arepolypeptides or proteins. Peptide libraries, e.g. combinatoriallibraries of peptides or other compounds can be fully randomized, withno sequence preferences or constants at any position. Alternatively, thelibrary can be biased, i.e., some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in some cases, the nucleotides or amino acidresidues are randomized within a defined class, for example, ofhydrophobic amino acids, hydrophilic residues, sterically biased (eithersmall or large) residues, towards the creation of cysteines, forcross-linking, prolines for SH-3 domains, serines, threonines, tyrosinesor histidines for phosphorylation sites, or to purines.

The candidate agents can also be nucleic acids. Nucleic acid candidateagents can be naturally occurring nucleic acids, random nucleic acids,or “biased” random nucleic acids. For example, digests of prokaryotic oreukaryotic genomes can be similarly used as described above forproteins.

In some embodiments, the candidate agent that is screened and identifiedto modulate viability, propagation and/or differentiation of apluripotent cell according to the methods described herein, can increaseviability, propagation and/or differentiation of a pluripotent cell byat least 5%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,10-fold, 50-fold, 100-fold or more relative to an untreated control. Insome embodiments, the candidate agent that is screened and identified tomodulate viability, propagation and/or differentiation of a pluripotentcell according to the methods described herein, can decrease viability,propagation and/or differentiation of a pluripotent cell by at least 5%,preferably at least 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 95%,97%, 98%, 99% or more, up to and including complete reduction (i.e.,zero viability, growth, propagation, or differentiation) relative to anuntreated control.

In some embodiments, the candidate agent functions directly in the formin which it is administered. Alternatively, the candidate agent can bemodified or utilized intracellularly to produce a form that modulatesthe desired activity, e.g. introduction of a nucleic acid sequence intoa cell and its transcription resulting in the production of an inhibitoror activator of gene expression or protein activity within the cell.

It is contemplated that the methods and compositions described hereincan be used, e.g. in the development of cancer vaccines. Generating atleast partially differentiated progeny of pluripotent tumor cellsobtained as described herein (e.g. by treating a mature tumor cell inaccordance with the methods described herein) can provide a diverse andchanging antigen profile which can permit the development of morepowerful APC (antigen presenting cells)-based cancer vaccines.

In some embodiments, the methods described herein relate to increasingthe transformation efficiency of a cell. Stressing cells, e.g., inducingpluripotency as described herein can make the cells more receptive tomethods of genetic modification including but not limited to transgeneinsertion, viral vectors, and/or zinc finger endonucleases. It iscontemplated that the methods described herein can permit cells to bemodified to a genetically receptive state such that naked DNA could beused to transform the resulting pluripotent cells.

Some aspects of the technology described herein relate to methods ofcell therapy comprising administering a pluripotent cell, produced bythe methods described herein, or the at least partially differentiatedprogeny of such a cell to a subject in need of cell therapy. In someembodiments, a therapeutically effective amount of pluripotent cells orthe at least partially differentiated progeny of the pluripotent cell isprovided. In some embodiments, the pluripotent cells and/or theirprogeny are autologous. In some embodiments, the pluripotent cellsand/or their progeny are allogeneic. In some embodiments, thepluripotent cells and/or their progeny are autologous. In someembodiments, the pluripotent cells and/or their progeny are HLA-matchedallogeneic. In some embodiments, the pluripotent cells and/or theirprogeny are syngeneic. In some embodiments, the pluripotent cells and/ortheir progeny are xenogeneic. In some embodiments, the cell therapy canbe autologous therapy, e.g. a cell from a subject can be used togenerate a pluripotent cell according to the methods described hereinand the pluripotent cell and/or at least partially differentiatedprogeny of that pluripotent cell can be administered to the subject. Asused herein, a “subject in need of cell therapy” refers to a subjectdiagnosed as having, or at risk of having or developing a diseaseassociated with the failure of a naturally occurring cell or tissue typeor a naturally occurring pluripotent and/or multipotent cell (e.g. stemcell).

In some embodiments, the methods described herein can be used to treatgenetic disorders, e.g. Tay-Sachs or hemophilia, e.g. by administeringallogeneic pluripotent cells and/or their progeny obtained as describedherein.

In one aspect, described herein is a method of preparing a cell ortissue that is compatible with cell therapy to be administered to asubject, comprising: generating a pluripotent cell (or more pluripotentcell) from a cell according to the methods described herein, wherein thecell is an autologous cell or HLA-matched allogeneic cell. In someembodiments, the pluripotent cell (or more pluripotent cell) can bedifferentiated along a pre-defined cell lineage prior to administeringthe cell or tissue to the subject.

Pluripotent cells, e.g. pluripotent stem cells, generated according tothe methods described herein can be used in cancer therapy. For example,high dose chemotherapy plus hematopoietic stem cell transplantation toregenerate the bone marrow hematopoietic system can benefit from the useof pluripotent cells generated as described herein.

Non-limiting examples of diseases associated with the failure of anaturally occurring cell or tissue type or a naturally occurringpluripotent and/or multipotent cell include aplastic anemia, Fanconianemia, and paroxysmal nocturnal hemoglobinuria (PNH). Others include,for example: acute leukemias, including acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (AML), acute biphenotypic leukemia andacute undifferentiated leukemia; chronic leukemias, including chronicmyelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), juvenilechronic myelogenous leukemia (JCML) and juvenile myelomonocytic leukemia(JMML); myeloproliferative disorders, including acute myelofibrosis,angiogenic myeloid metaplasia (myelofibrosis), polycythemia vera andessential thrombocythemia; lysosomal storage diseases, includingmucopolysaccharidoses (MPS), Hurler's syndrome (MPS-IH), Scheie syndrome(MPS-IS), Hunter's syndrome (MPS-II), Sanfilippo syndrome (MPS-III),Morquio syndrome (MPS-IV), Maroteaux-Lamy Syndrome (MPS-VI), Slysyndrome, beta-glucuronidase deficiency (MPS-VII), adrenoleukodystrophy,mucolipidosis II (I-cell Disease), Krabbe disease, Gaucher's disease,Niemann-Pick disease, Wolman disease and metachromatic leukodystrophy;histiocytic disorders, including familial erythrophagocyticlymphohistiocytosis, histiocytosis-X and hemophagocytosis; phagocytedisorders, including Chediak-Higashi syndrome, chronic granulomatousdisease, neutrophil actin deficiency and reticular dysgenesis; inheritedplatelet abnormalities, including amegakaryocytosis/congenitalthrombocytopenia; plasma cell disorders, including multiple myeloma,plasma cell leukemia, and Waldenstrom's macroglobulinemia. Othermalignancies treatable with stem cell therapies include but are notlimited to breast cancer, Ewing sarcoma, neuroblastoma and renal cellcarcinoma, among others. Also treatable with stem cell therapy are: lungdisorders, including COPD and bronchial asthma; congenital immunedisorders, including ataxia-telangiectasia, Kostmann syndrome, leukocyteadhesion deficiency, DiGeorge syndrome, bare lymphocyte syndrome,Omenn's syndrome, severe combined immunodeficiency (SCID), SCID withadenosine deaminase deficiency, absence of T & B cells SCID, absence ofT cells, normal B cell SCID, common variable immunodeficiency andX-linked lymphoproliferative disorder; other inherited disorders,including Lesch-Nyhan syndrome, cartilage-hair hypoplasia, Glanzmannthrombasthenia, and osteopetrosis; neurological conditions, includingacute and chronic stroke, traumatic brain injury, cerebral palsy,multiple sclerosis, amyotrophic lateral sclerosis and epilepsy; cardiacconditions, including atherosclerosis, congestive heart failure andmyocardial infarction; metabolic disorders, including diabetes; andocular disorders including macular degeneration and optic atrophy. Suchdiseases or disorders can be treated either by administration ofpluripotent cells themselves, permitting in vivo differentiation to thedesired cell type with or without the administration of agents topromote the desired differentiation, and/or by administering pluripotentcells differentiated to, or at least partially differentiated towardsthe desired cell type in vitro. Methods of diagnosing such conditionsare well known to medical practitioners of ordinary skill in the art. Insome embodiments, the subject can be one who was treated with radiationtherapy or other therapies which have ablated a population of cells orstem cells, e.g. the subject can be a subject with cancer whose bonemarrow has been ablated by radiation therapy.

In some embodiments, pluripotent cells are administered to the subject.In some embodiments, an at least partially differentiated cell isadministered to the subject. In some embodiments, the method of celltherapy can further comprise differentiating the pluripotent cell alonga pre-defined cell lineage prior to administering the cell. Methods ofdifferentiating stem cells along desired cell lineages are known in theart and examples are described herein.

In some embodiments, a composition comprising a pluripotent cellobtained according to the methods described herein or an at leastpartially differentiated cell which is the progeny of the pluripotentcell is administered to the subject.

In some embodiments, a composition comprising a pluripotent cellobtained according to the methods described herein, or an at leastpartially differentiated cell which is the progeny of the pluripotentcell, can optionally further comprise G-CSF, GM-CSF and/or M-CSF and/orcan be administered to a subject who has or will be administered G-CSF,GM-CSF and/or M-CSF in a separate composition. Administration of G-CSF,GM-CSF and/or M-CSF can, e.g. induce a state of inflammation favorableto organ regeneration and removal of tissue debris, waste and buildup.

In some embodiments, administration of the pluripotent cells and/ortheir at least partially differentiated progeny can occur within arelatively short period of time following production of the pluripotentcell in culture according to the methods described herein (e.g. 1, 2, 5,10, 24 or 48 hours after production). In some embodiments,administration of the at least partially differentiated progeny canoccur within a relatively short period of time following differentiationof the pluripotent cell in culture according to the methods describedherein (e.g. 1, 2, 5, 10, 24 or 48 hours after production). In someembodiments, the pluripotent cells and/or their at least partiallydifferentiated progeny can be cryogenically preserved prior toadministration.

In some aspects, the technology described herein relates to acomposition comprising a pluripotent cell generated according to themethods described herein and/or the at least partially differentiatedprogeny of the pluripotent cell. In some embodiments, a pharmaceuticalcomposition comprises a pluripotent cell generated according to themethods described herein and/or the at least partially differentiatedprogeny of the pluripotent cell, and optionally a pharmaceuticallyacceptable carrier. The compositions can further comprise at least onepharmaceutically acceptable excipient.

The pharmaceutical composition can include suitable excipients, orstabilizers, and can be, for example, solutions, suspensions, gels, oremulsions. Typically, the composition will contain from about 0.01 to 99percent, preferably from about 5 to 95 percent of cells, together withthe carrier. The cells, when combined with pharmaceutically orphysiologically acceptable carriers, excipients, or stabilizer, can beadministered parenterally, subcutaneously, by implantation or byinjection. For most therapeutic purposes, the cells can be administeredvia injection as a solution or suspension in liquid form. The term“pharmaceutically acceptable carrier” refers to a carrier foradministration of the pluripotent cell generated according to themethods described herein and/or the at least partially differentiatedprogeny of the pluripotent cell. Such carriers include, but are notlimited to, saline, buffered saline, dextrose, water, glycerol, andcombinations thereof. Each carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation, forexample the carrier does not decrease the impact of the agent on thesubject. In other words, a carrier is pharmaceutically inert andcompatible with live cells.

Suitable formulations also include aqueous and non-aqueous sterileinjection solutions which can contain anti-oxidants, buffers,bacteriostats, bactericidal antibiotics and solutes which render theformulation isotonic with the bodily fluids of the intended recipient.Aqueous and non-aqueous sterile suspensions can include suspendingagents and thickening agents. The formulations can be presented inunit-dose or multi-dose containers.

Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, suspensions ready for injection, andemulsions. Parenteral dosage forms can be prepared, e.g., usingbioresorbable scaffold materials to hold pluripotent cells generatedaccording to the methods described herein and/or the at least partiallydifferentiated progeny of the pluripotent cell.

The term ‘epigenetic modification’ refers to the chemical marking of thegenome. Epigenetic marks can include DNA methylation (imprints) as wellas methylation and acetylation of proteins associated with DNA, such ashistories. Parent-of-origin-specific gene expression (either from thematernal or paternal chromosome) is often observed in mammals and is dueto epigenetic modifications. In the parental germlines, epigeneticmodification can lead to stable gene silencing or activation.

As used herein, the term “administer” or “transplant” refers to theplacement of cells into a subject by a method or route which results inat least partial localization of the cells at a desired site such that adesired effect is produced.

The pluripotent stem cells described herein, and/or their at leastpartially differentiated progeny, can be administered in any mannerfound appropriate by a clinician and can include local administration,e.g. by injection of a suspension of cells or, for example, byimplantation of a preparation of cells deposited or grown on or withinan implantable scaffold or support. Implantable scaffolds can includeany of a number of degradable or resorbable polymers, or, for example, asilk scaffold, among others. Suitable routes for administration of apharmaceutical composition comprising pluripotent stem cells describedherein, and/or their at least partially differentiated progeny includebut are not limited to local administration, e.g. intraperitoneal,parenteral, intracavity or subcutaneous administration. The phrases“parenteral administration” and “administered parenterally” as usedherein, refer to modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intraperitoneal, intradermal, subcutaneous injection and infusion.Administration can involve the use of needles, catheters and syringessuitable for injection, or surgical implantation. The use of acombination of delivery means and sites of delivery are contemplated toachieve the desired clinical effect.

The term ‘epigenetic modification’ refers to the chemical marking of thegenome. Epigenetic marks can include DNA methylation (imprints) as wellas methylation and acetylation of proteins associated with DNA, such ashistones. Parent-of-origin-specific gene expression (either from thematernal or paternal chromosome) is often observed in mammals and is dueto epigenetic modifications. In the parental germlines, epigeneticmodification can lead to stable gene silencing or activation.

In one embodiment, a therapeutically effective amount of pluripotentstem cells described herein, and/or their at least partiallydifferentiated progeny is administered to a subject. A “therapeuticallyeffective amount” is an amount of pluripotent stem cells describedherein, and/or their at least partially differentiated progeny,sufficient to produce a measurable improvement in a symptom or marker ofthe condition being treated. Actual dosage levels of cells in atherapeutic composition can be varied so as to administer an amount ofthe cells that is effective to achieve the desired therapeutic responsefor a particular subject. The selected dosage level will depend upon avariety of factors including, but not limited to, the activity of thetherapeutic composition, formulation, the route of administration,combination with other drugs or treatments, severity of the conditionbeing treated, the physical condition of the subject, prior medicalhistory of the subject being treated and the experience and judgment ofthe clinician or practitioner administering the therapy. Generally, thedose and administration scheduled should be sufficient to result inslowing, and preferably inhibiting progression of the condition and alsopreferably causing a decrease in one or more symptoms or markers of thecondition. Determination and adjustment of a therapeutically effectivedose, as well as evaluation of when and how to make such adjustments,are known to those of ordinary skill in the art of medicine.

The dosage of pluripotent stem cells described herein, and/or their atleast partially differentiated progeny administered according to themethods described herein can be determined by a physician and adjusted,as necessary, to suit observed effects of the treatment. With respect toduration and frequency of treatment, it is typical for skilledclinicians to monitor subjects in order to determine when the treatmentis providing therapeutic benefit, and to determine whether to administeranother dose of cells, increase or decrease dosage, discontinuetreatment, resume treatment, or make other alteration to the treatmentregimen. Where cells administered are expected to engraft and survivefor medium to long term, repeat dosages can be necessary. However,administration can be repeated as necessary and as tolerated by thesubject. The dosage should not be so large as to cause substantialadverse side effects. The dosage can also be adjusted by the individualphysician in the event of any complication. Typically, however, thedosage can range from 100 to 1×10⁹ pluripotent stem cells as describedherein, and/or their at least partially differentiated progeny for anadult human, e.g. 100 to 10,000 cells, 1,000 to 100,000 cells, 10,000 to1,000,000 cells, or 1,000,000 to 1×10⁹ cells. Effective doses can beextrapolated from dose-response curves derived from, for example, animalmodel test bioassays or systems.

Therapeutic compositions comprising pluripotent stem cells describedherein, and/or their at least partially differentiated progeny preparedas described herein are optionally tested in one or more appropriate invitro and/or in vivo animal models of disease, such as a SCID mousemodel, to confirm efficacy, evaluate in vivo growth of the transplantedcells, and to estimate dosages, according to methods well known in theart. In particular, dosages can be initially determined by activity,stability or other suitable measures of treatment vs. non-treatment(e.g., comparison of treated vs. untreated animal models), in a relevantassay. In determining the effective amount of pluripotent stem cellsdescribed herein, and/or their at least partially differentiatedprogeny, the physician evaluates, among other criteria, the growth andvolume of the transplanted cells and progression of the condition beingtreated. The dosage can vary with the dosage form employed and the routeof administration utilized.

With respect to the therapeutic methods described herein, it is notintended that the administration of pluripotent stem cells describedherein, and/or their at least partially differentiated progeny belimited to a particular mode of administration, dosage, or frequency ofdosing. All modes of administration are contemplated, includingintramuscular, intravenous, intraperitoneal, intravesicular,intraarticular, intralesional, subcutaneous, or any other routesufficient to provide a dose adequate to treat the condition beingtreated.

In some embodiments, the methods described herein can be used togenerate pluripotent cells in vivo, e.g. a cell present in a subject canbe subjected to a stress as described herein such that acquires apluripotent phenotype. Methods of applying the stresses described hereinto cells in vivo are readily apparent, e.g. mild acid solutions can beintroduced to a tissue via injection and/or direct application,temperatures can be altered by probes which can heat or cool thesurrounding tissue or via the use of non-invasive methods, e.g. focusbeam radiation. In vivo modulation of pluripotency can be used to, e.g.increase tissue regeneration or wound healing. Non-limiting examples caninclude the injection of a mild acid into an arthritic knee joint toinduce knee joint cells (e.g. synovial or cartilage cells) to assume apluripotent phenotype and generate new tissues. A further non-limitingexample can include the treatment of a subject with a stroke or centralnervous system injury (e.g. spinal cord injury). After inflammation hasresolved, the cells adjacent to the injured area can be treated with astress as described herein, generating pluripotent cells that canrepopulate the damaged tissue and/or regenerate or repair the damagedtissue.

In a further non-limiting example, changes in epigenetic status (e.g. bytreatment with a demethylase) can cause non-insulin secreting cells(e.g. alpha glugagon cells of the pancreas) to convert toinsulin-secreting cells (e.g. beta cells). Accordingly, treating anon-insulin secreting cell (e.g. an alpha glugagon cell of the pancreas)in accordance with the methods described herein can result in the cellbecoming an insulin-secreting cell, e.g. a beta-like cell, either invivo or in vitro.

Further, it is contemplated that the pluripotent cells described hereincan fuse with other cells (i.e. “recipient cells”), e.g. cells nottreated according to the methods described herein, non-pluripotentcells, mature cells, malignant cells, and/or damaged cells. The fusionof the cells can result in an increased level of cellular repair enzymeexpression and/or activity in the recipient cell as compared to prior tothe fusion. This can increase the health and/or function of therecipient cell, e.g. by increasing repair of cellular damage, mutations,and/or modification of the epigenetic status of the recipient cell.

In some embodiments, by increasing the pluripotency of cells in vivo,the epigenetic markers (e.g. DNA methylation, demethylation, and/orhydroxymethylation status) of those cells can be modulated. Modulationof epigenetic markers has been implicated in, e.g. malignancy,arthritis, autoimmune disease, aging, etc and the treatment of suchepigenetically-linked conditions in accordance with the methodsdescribed herein is contemplated.

In some embodiments, multiple tissues can be treated in vivo at the sametime, e.g. a mildly acidic state could be induced in multiple organs,e.g. successively or in synchrony (e.g. brain, heart, liver, lung,and/or thyroid) to treat widespread damage or aging.

It is further contemplated that the in vivo treatment of cells asdescribed herein can be combined with the administration of pluripotentcells and/or the at least partially differentiated progeny thereof whichhave been produced as described herein.

It is contemplated herein that the methods described herein can be usedto treat, e.g. a fetus or embryo in utero.

Efficacy of treatment can be assessed, for example by measuring amarker, indicator, symptom or incidence of, the condition being treatedas described herein or any other measurable parameter appropriate, e.g.number of pluripotent cell progeny. It is well within the ability of oneskilled in the art to monitor efficacy of treatment or prevention bymeasuring any one of such parameters, or any combination of parameters.

Effective treatment is evident when there is a statistically significantimprovement in one or more markers, indicators, or symptoms of thecondition being treated, or by a failure to worsen or to developsymptoms where they would otherwise be anticipated. As an example, afavorable change of at least about 10% in a measurable parameter of acondition, and preferably at least about 20%, about 30%, about 40%,about 50% or more can be indicative of effective treatment. Efficacy forpluripotent cells generated according to the methods described hereinand/or the at least partially differentiated progeny of the pluripotentcell can also be judged using an experimental animal model known in theart for a condition described herein. When using an experimental animalmodel, efficacy of treatment is evidenced when a statisticallysignificant change in a marker is observed, e.g. the number ofhematopoietic cells present in a mouse following bone marrow ablationand treatment with pluripotent cells as described herein.

In one aspect, described herein is a method of producing a pluripotentcell capable of differentiating into a placental cell, the methodcomprising culturing a pluripotent cell obtained according to themethods described herein in the presence of FGF4. In some embodiments,the pluripotent cell is capable of differentiating into an embryonicstem cell. In some embodiments, the concentration of FGF4 is from about1 nM to about 1 uM. In some embodiments, the concentration of FGF4 isfrom 1 nM to 1 uM. In some embodiments, the concentration of FGF4 isfrom about 5 nM to about 500 nM. In some embodiments, the concentrationof FGF4 is from about 10 nM to about 100 nM.

In some aspects, the technology described herein relates to a system forgenerating a pluripotent cell from a cell, comprising removing a portionof the cytoplasm and/or mitochondria from the cell.

A system for generating a pluripotent cell from a cell, according to themethods described herein, can comprise a container in which the cellsare subjected to stress. The container can be suitable for culture ofsomatic and/or pluripotent cells, as for example, when cells arecultured for days or longer under low oxygen conditions in order toreduce the amount of cytoplasm and/or mitochondria according to themethods described herein. Alternatively, the container can be suitablefor stressing the cells, but not for culturing the cells, as forexample, when cells are triturated in a device having a narrow aperturefor a limited period, e.g. less than 1 hour. A container can be, forexample, a vessel, a tube, a microfluidics device, a pipette, abioreactor, or a cell culture dish. A container can be maintained in anenvironment that provides conditions suitable for the culture of somaticand/or pluripotent cells (e.g. contained within an incubator) or in anenvironment that provides conditions which will cause environmentalstress on the cell (e.g. contained within an incubator providing a lowoxygen content environment). A container can be configured to provide 1or more of the environmental stresses described above herein, e.g. 1stress, 2 stresses, 3 stresses, or more. Containers suitable formanipulation and/or culturing somatic and/or pluripotent cells are wellknown to one of ordinary skill in the art and are available commercially(e.g. Cat No CLS430597 Sigma-Aldrich; St. Louis, Mo.). In someembodiments, the container is a microfluidics device. In someembodiments, the container is a cell culture dish, flask, or plate.

In some embodiments, the system can further comprise a means forselecting pluripotent cells, e.g. the system can comprise a FACS systemwhich can select cells expressing a pluripotency marker (e.g. Oct4-GFP)or select by size as described above herein. Methods and devices forselection of cells are well known to one of ordinary skill in the artand are available commercially, e.g. BD FACSARIA SORP™ coupled with BDLSRII™ and BD FACSDIVA™ Software (Cat No. 643629) produced by BDBiosciences; Franklin Lakes, N.J.

In some embodiments, cells which are not present in a tissue areprovided to the system. In some embodiments, tissues are provided to thesystem and the system further comprises a means of isolating one or moretypes of cells. By way of non-limiting example, the system can comprisea tissue homogenizer. Tissue homogenizers and methods of using them areknown in the art and are commercially available (e.g. FASTH21™, Cat No.21-82041 Omni International; Kennesaw, Ga.). Alternatively, the systemcan comprise a centrifuge to process blood or fluid samples.

In some embodiments, the system can be automated. Methods of automatingcell isolation, cell culture, and selection devices are known in the artand are commercially available. For example, the FASTH21™ TissueHomogenizer (Cat No. 21-82041 Omni International; Kennesaw, Ga.) and theBD FACSARIA SORP™.

In some embodiments, the system can be sterile, e.g. it can be operatedin a sterile environment or the system can be operated as a closed,sterile system.

In one aspect, described herein is a method of increasing theself-renewal ability of a pluripotent cell, the method comprisingculturing the cell in the presence of adrenocorticotropic hormone(ACTH), 2i or 3i medium. As used herein, “self-renewal ability” refersto the length of time a cell can be cultured and passaged in vitro, e.g.the number of passages a cell and it's progeny can be subjected to andcontinue to produce viable cells. The cell which is caused to have anincreased self-renewal ability according to the method described hereincan be, e.g. a totipotent cell and/or a cell generated by exposing it tostress as described elsewhere herein.

In some embodiments, culturing in the presence of ACTH can compriseculturing the cell in a cell medium comprising from about 0.1 μM toabout 1,000 μM, e.g. from about 0.1 μM to about 100 μM, from about 0.1μM to about 10 μM, or about 10 μM. In some embodiments, culturing thecell in the presence of ACTH can comprise culturing the cell in LIFmedium comprising ACTH. LIF, ACTH, 2i and 3i are commercially availableand well known in the art, e.g. ACTH can be purchased from Sigma-Aldrich(Cat No. A0673; St. Louis, Mo.) and LIF media can be purchased fromMillipore (e.g. Cat Nos ESG1107; Billerica, Mass.), and 3i can bepurchased from Stem Cells Inc. (e.g. as “iSTEM Stem Cell Culture Medium,Cat No. SCS-SF-ES-01; Newark, Calif.).

In some embodiments, the culturing step can proceed for at least 3 days,e.g. at least 3 days, at least 4 days, at least 5 days, at least 6 days,at least 7 days, or longer. After the culturing step, the cells can bemaintained under conditions suitable for maintaining pluripotent cellsas described elsewhere herein.

In some embodiments, after the culturing step, the cell can express adetectable and/or increased level of a stem cell marker. Stem cellmarkers and methods of detecting them are described elsewhere herein. Insome embodiments, the stem cell marker can be selected from the groupconsisting of Oct3/4; Nanog; Rex1; Klf4; Sox2; Klf2; Esrr-beta; Tbx3;and Klf5.

In one aspect, provided herein are methods for generating pluripotent orSTAP cells that is an improvement over the preceeding methods, e.g.provides increased efficiency, quality and/or yield.

In one embodiment, provided herein is a method for generatingpluripotent or STAP cells from, e.g., a cell suspension and/or tissueculture conditions.

As a first step, the initial (e.g. starting material) cell in suspensioncan be pelleted and/or removed from solution. As but one example, thecell can be pelleted by centrifugation in a centrifuge tube for fromabout 800 rpm to about 1600 rpm for from about 1 minute to about 20minutes. As but one example, the cell can be pelleted by centrifugationin a centrifuge tube for about 1200 rpm for 5 minutes. In someembodiments, the cell in suspension can be contacted with digestiveenzyme such as trypsin prior to being pelleted and/or removed fromsolution. As but one example, Trypsin-EDTA, at from about 0.01% to about0.5% (Gibco: 25300-054) can be added to a tissue culture dish containingcells, for from about 1 to about 20 minutes, to release adherent cellsto be added to a centrifuge tube. As but one example, Trypsin-EDTA,0.05% (Gibco: 25300-054) can be added to a tissue culture dishcontaining cells, for from about 3-5 minutes, to release adherent cellsto be added to a centrifuge tube. In embodiments comprisingcentrifugation, the supernatant can be aspirated down to the cell pelletfollowing centrifugation.

In some embodiments, the first step is performed on a population of atleast 1 million viable cells. In some embodiments, the first step isperformed on a population of at least 5 million viable cells. In someembodiments, the first step is performed on a population of at least 10million viable cells.

As a second step, the cells can be resuspended in physiological saline,e.g., HBSS (Hanks Balanced Saline Solution) (e.g., HBSS Ca⁺Mg⁺ Free:Gibco 14170-112). In some embodiments, the cell can be resuspended at aconcentration of from about 1×10³ cells/mL to about 1×10⁹ cells/mL. Insome embodiments, the cell can be resuspended at a concentration of fromabout 1×10⁵ cells/mL to about 1×10⁷ cells/mL In some embodiments, thecell can be resuspended at a concentration of about 1×10⁶ cells/mL. Insome embodiments, the cells can be resuspended in a 50 mL tube. In someembodiments, the cells can be resuspended in 2-3 mL HBSS in a 50 mLtube.

As a third step, the cell, in suspension/solution, can be triturated,e.g. passed through an aperture, opening, and/or lumen sufficientlysmall to generate, e.g. shear stresses. In exemplary embodimentsdescribed below herein, the aperture, opening, and/or lumen is comprisedby a glass pipette having an opening of a size as described belowherein. Trituration can be accomplished by a number of alternativemeans. Non-limiting examples can include apertures, lumens, or channelsin a microfluidics device, a cell-handling device having a pump andtubing, passing a cell suspension through a grate or filter, causing acell suspension to flow past barriers or particles, and the like, One ofskill in the art can empirically determine the appropriate pressures,flow rates, shear stress, etc for different trituation systems basedupon the present disclosure. Further discussion of fluid stresses andcalculations relevant to such stresses can be found in e.g., Fournier“Basic Transport Phenomena in Biomedical Engineering” Taylor & Francis,1999; which is incorporated by reference herein in its entirety.

In some embodiments, the trituration can last for from about 10 minutesto about 2 hours, e.g. from about 20 minutes to about 1 hours, or about30 minutes. In some embodiments, the trituration can last for at least10 minutes, e.g. 10 minutes or more, 20 minutes or more, 30 minutes ormore, 40 minutes or more, 50 minutes or more, or 60 minutes or more. Insome embodiments, the trituration can continue until the suspension canbe easily triturated through the opening or lumen. In some embodiments,the trituration in the last aperture or lumen can be continued until thesuspension passes easily through the aperture or lumen. In someembodiments, the trituration in each aperture or lumen can be continueduntil the suspension passes easily through that aperture or lumen.

In some embodiments, the trituration can comprise trituration through aseries of openings or lumens, e.g. a series of progressively smalleropenings or lumens. In some embodiments, the series of openings orlumens comprises at least 2 openings or lumens, e.g. 2, 3, 4, 5, 10, 20,50, or more openings or lumens. In some embodiments, one or more of theopenings or lumens can be pre-coated, e.g. with HBSS or water.

As but an exemplary embodiment, the cells can be triturated throughmultiple, e.g., three openings or lumens. In some embodiments, the firstopening or lumen can have an internal diameter of from about 0.5 mm toabout 2.0 mm. In some embodiments, the first opening or lumen can havean internal diameter of from about 0.7 mm to about 1.5 mm. In someembodiments, the first opening or lumen can have an internal diameter ofabout 1.1 mm. In some embodiments, the trituration through the firstaperture or lumen can be performed for from about 1 minute to about 10minutes. In some embodiments, the trituration through the first apertureor lumen can be performed for about 5 minutes. As but an exemplaryembodiment, the first opening or lumen is comprised by a standard 9″glass pipette (e.g., Fisher brand 9″ Disposable Pasteur Pipettes:13-678-20D) and the cell suspension can be triturated in and out of thepipette for 5 minutes with a fair amount of force to dissociate cellaggregates and any associated debris.

In some embodiments, the last two apertures or lumens in the series canhave internal diameters of from about 90 to about 200 microns and fromabout 25 microns to about 90 microns. In some embodiments, the last twoapertures or lumens in the series can have internal diameters of fromabout 100 to about 150 microns and from about 50 microns to about 70microns. In some embodiments, the trituration can comprise about 5 toabout 20 minutes of trituration through the second to last aperture orlumen and about 5 to about 20 minutes of trituration in the lastaperture or lumen. In some embodiments, the trituration can comprisesabout 10 minutes of trituration through the second to last aperture orlumen and about 15 minutes of trituration in the last aperture or lumen.

As but an exemplary embodiment, the last two openings or lumens can becomprised by pipettes modified as follows: Make two fire polishedpipettes with very small orifices as follows: Heat the standard 9″ glasspipette over, e.g., a Bunsen burner and then pull and stretch the distal(melting) end of the pipette, until the lumen collapses and the tipbreaks off, leaving a closed, pointed glass tip. Wait until the pipettecools, and then break off the closed distal tip until a very small lumenis now identifiable. Repeat this process with the second pipette, butbreak the tip off a little more proximally, creating a slightly largerdistal lumen. The larger lumen should be about 100-150 microns indiameter, while the other pipette should have a smaller lumen of about50-70 microns. The cell suspension can be triturated through the pipettewith the larger lumen for 10 minutes. This can be followed withtrituration through the pipette having the smaller lumen (50-70 microns)for an additional 15 minutes. Continue to triturate the suspension untilit passes easily up and down the fire polished pipette of the smallerbore. Each pipette can be precoated with media. Also, duringtrituration, aspirating air and creating bubbles or foam in the cellsuspension is to be avoided.

In some embodiments, trituration can be performed at a rate of fromabout 1 to about 200 cycles per minute, e.g. the entire suspension ispassed through an aperture, lumen, or opening 1 to 100 times per minute.In some embodiments, trituration can be performed at a rate of fromabout 10 to about 60 cycles per minute. In some embodiments triturationcan be performed at a rate of about 40 cycles per minute. In someembodiments, wherein a pipette is used for trituration, the suspensioncan be passed out of and back into the pipette about 20 times perminute.

In a next step, the triturated cells can be isolated from thesuspension. In some embodiments, about 0 to 50 volumes of HBSS can beadded to the triturated suspension and the suspension centrifuged atfrom about 800-1600 rpm for from about 1 minute to about 30 minutesminutes and then the supernatant aspirated. In some embodiments, about 9volumes of HBSS can be added to the triturated suspension and thesuspension centrifuged at about 1200 rpm for about 5 minutes minutes andthen the supernatant aspirated.

In a next step, the cells can be resuspended in HBSS, with the resultingsuspension having a pH of from about 5.0 to about 6.0. In someembodiments, the resulting suspension can have a pH of from about 5.4 toabout 5.8. In some embodiments, the resulting suspension can have a pHof from about 5.6 to about 5.7. In some embodiments, the resultingsuspension can have a pH of about 5.6. In some embodiments, the HBSSsolution prior to admixture with the cells can have a pH of from about5.0 to about 5.7. In some embodiments, the HBSS solution prior toadmixture with the cells can have a pH of from about 5.3 to about 5.6.In some embodiments, the HBSS solution prior to admixture with the cellscan have a pH of about 5.4. In some embodiments, the cells can beresuspended at a concentration of from about 2×10⁴ cells/mL to about2×10⁸ cells/mL. In some embodiments, the cells can be resuspended at aconcentration of about 2×10⁶.

As but an exemplary example the resuspension step of the preceedingparagraph can be performed as follows: when making the solution acidic,mildly pipette it using a 5 ml pipette for 10 seconds immediately afteradding the acid to the Hanks Solution. HBSS has a very weak bufferingcapacity, so any solution transferred from the supernatant of theprevious suspension will affect the pH of the HBSS drastically. Theinstructions below will show how to create HBSS with the optimum pH of5.6-5.7 for STAP cell generation according to this experimentalembodiment. First, titrate the pH of pre-chilled HBSS (at 4 degrees C.)with 12N HCl to a pH of 5.6. This is done by slowly adding 11.6 ul of 12N HCl to 50 ml of HBSS. After confirming this pH, sterilize the solutionby filtering through a 0.2 micron syringe filter or bottle top filterof, into a new sterile container for storage. Please confirm, forexample, the final pH of 5.6-5.7 through an initial test experiment withan appropriate number of cells. Because the pH of the HBSS is soimportant, the pH of the solution be checked, re-titrated andre-sterilized prior to each use.

In a next step, the cells in the HBSS suspension can be incubated atabout their in vivo temperature. For example, mammalian cells can beincubated at about 37° C. In some embodiments, the incubation can be forfrom about 5 minutes to about 3 hours. In some embodiments, theincubation can be for from about 10 minutes to about 1 hour. In someembodiments, the incubation can be for from about 15 minutes to about 40minutes. In some embodiments, the incubation can be for about 25minutes.

In a next step, the cells are isolated from the acidic HBSS solution. Asbut one example, the cells can be pelleted by centrifugation in acentrifuge tube for from about 800 rpm to about 1600 rpm for from about1 minute to about 20 minutes. As but one example, the cells can bepelleted by centrifugation in a centrifuge tube for about 1200 rpm for 5minutes. In some embodiments, the supernatant can then be aspirated.

In a next step, the cells can be resuspended in media suitable formaintaining and/or selecting a pluripotent cell. In some embodiments,the media is sphere media. As used herein, “sphere media refers toDMEM/F12 with 1% Antibiotic and 2% B27 Gibco 12587-010. In someembodiments, the media can further comprise growth factors, e.g., b-FGF(20 ng/ml), EGF (20 ng/ml), heparin (0.2%, Stem Cell Technologies07980). These factors are tailored to the type of cell used. Forexample, In some embodiments, LIF (1000 U) can be added if the cells aremurine). In some embodiments, supplements such as bFGF, EGF and heparinmay not be necessary.) In some embodiments, the cells can be resuspendedin media at a concentration of 10⁵ cells/cc.

In a next step, the cells can be cultured and/or maintained, e.g.cultured at 37° C. with 5% CO₂. In some embodiments, the cells can beagitated during culturing/maintaining to prevent adherence to a cellculture container. In some embodiments, the cells can be gently pipettedusing, for example, a 5 ml pipette, twice/day for 2 minutes, for thefirst week, to discourage them from attaching to the bottom of thedishes. In some embodiments, this can promote good sphere formation. Insome embodiments, sphere media, optionally containing supplements, canbe added every other day. For example, add 1 ml/day to a 10 cm culturedish, or 0.5 ml/day to a 6 cm dish.

In a second embodiment, provided herein is a method for generatingpluripotent or STAP cells from, e.g., a soft tissue that may comprisered blood cells (RBCs). Such tissues can include, but are not limited tothe liver, spleen, and lung.

In a first step, the soft tissue, (e.g. an excised, washed, sterileorgan tissue) is mechanically sliced, minced scraped, and/or macerated.In some embodiments, this step can be performed in the presence ofdigestive enzymes and/or enzymes that degrade the ECM. In someembodiments, the enzyme can be collagenase. It is contemplated hereinthat different types of collagenase or enzymes are better for digestionof different organ tissues, based upon the components of that tissue'sECM and connective tissues. One of skill in the art can readilydetermine appropriate enzymes for each tissue type. In some embodiments,the tissue is spleen and no enzyme is necessary. As but an exemplaryexample, the tissue can be minced and scraped for from about 1 minute toabout 30 minutes using scalpels and/or scissors to increase surface areathat is exposed to the collagenase, until the tissue appears to becomegelatinous in consistency. As but an exemplary example, the tissue canbe minced and scraped for from about 10 minutes using scalpels and/orscissors. In some embodiments, additional enzyme can be added and thetissue incubated with the enzyme, optionally with agitation. As but oneexample, the tissue can be kept in an incubator/shaker for 30 minutes at37° C. at 90 RPM. In some embodiments, the tissue can be diluted in HBSSafter enzyme exposure and/or mechanical disruption.

In a next step, the cell, in suspension, can be triturated, e.g. passedthrough an opening or lumen sufficiently small to generate, e.g. shearstresses. In some embodiments, the trituration can last for from about10 minutes to about 2 hours, e.g. from about 20 minutes to about 1hours, or about 30 minutes. In some embodiments, the trituration canlast for at least 10 minutes, e.g. 10 minutes or more, 20 minutes ormore, 30 minutes or more, 40 minutes or more, 50 minutes or more, or 60minutes or more. In some embodiments, the trituration can continue untilthe suspension can be easily triturated through the opening or lumen. Insome embodiments, the trituration in the last aperture or lumen can becontinued until the suspension passes easily through the aperture orlumen. In some embodiments, the trituration in each aperture or lumencan be continued until the suspension passes easily through thataperture or lumen.

In some embodiments, the trituration can comprise trituration through aseries of openings or lumens, e.g. a series of progressively smalleropenings or lumens. In some embodiments, the series of openings orlumens comprises at least 2 openings or lumens, e.g. 2, 3, 4, 5, 10, 20,50, or more openings or lumens. In some embodiments, one or more of theopenings or lumens can be pre-coated, e.g. with HBSS or water.

As but an exemplary embodiment the cells can be triturated through threeopenings or lumens. In some embodiments, the first opening or lumen canhave an internal diameter of from about 0.5 mm to about 2.0 mm. In someembodiments, the first opening or lumen can have an internal diameter offrom about 0.7 mm to about 1.5 mm. In some embodiments, the firstopening or lumen can have an internal diameter of about 1.1 mm. In someembodiments, the trituration through the first aperture or lumen can beperformed for from about 1 minute to about 10 minutes. In someembodiments, the trituration through the first aperture or lumen can beperformed for about 5 minutes. As but an exemplary embodiment, the firstopening or lumen is comprised by a standard 9″ glass pipette (e.g.,Fisher brand 9″ Disposable Pasteur Pipettes: 13-678-20D) and the cellsuspension can be triturated in and out of the pipette for 5 minuteswith a fair amount of force to dissociate cell aggregates and anyassociated debris.

In some embodiments, the last two apertures or lumens in the series canhave internal diameters of from about 90 to about 200 microns and fromabout 25 microns to about 90 microns. In some embodiments, the last twoapertures or lumens in the series can have internal diameters of fromabout 100 to about 150 microns and from about 50 microns to about 70microns. In some embodiments, the trituration can comprise about 5 toabout 20 minutes of trituration through the second to last aperture orlumen and about 5 to about 20 minutes of trituration in the lastaperture or lumen. In some embodiments, the trituration can comprisesabout 10 minutes of trituration through the second to last aperture orlumen and about 15 minutes of trituration in the last aperture or lumen.

As but an exemplary embodiment, the last two openings or lumens can becomprised by pipettes modified as follows: Make two fire polishedpipettes with very small orifices as follows: Heat the standard 9″ glasspipette over a Bunsen burner and then pull and stretch the distal(melting) end of the pipette, until the lumen collapses and the tipbreaks off, leaving a closed, pointed glass tip. Wait until the pipettecools, and then break off the closed distal tip until a very small lumenis now identifiable. Repeat this process with the second pipette, butbreak the tip off a little more proximally, creating a slightly largerdistal lumen. The larger lumen should be about 100-150 microns indiameter, while the other pipette should have a smaller lumen of about50-70 microns. The cell suspension can be triturated through the pipettewith the larger lumen for 10 minutes. This can be followed withtrituration through the pipette having the smaller lumen (50-70 microns)for an additional 15 minutes. Continue to triturate the suspension untilit passes easily up and down the fire polished pipette of the smallerbore. Each pipette can be precoated with media. Also, duringtrituration, aspirating air and creating bubbles or foam in the cellsuspension is to be avoided.

In some embodiments, trituration can be performed at a rate of fromabout 1 to about 200 cycles per minute, e.g. the entire suspension ispassed through an aperture, lumen, or opening 1 to 100 times per minute.In some embodiments, trituration can be performed at a rate of fromabout 10 to about 60 cycles per minute. In some embodiments triturationcan be performed at a rate of about 40 cycles per minute. In someembodiments, wherein a pipette is used for trituration, the suspensioncan be passed out of and back into the pipette about 20 times perminute.

In a next step, the non-RBC triturated cells can be isolated from redblood. In some embodiments, about 0 to 50 volumes of HBSS can be addedto the triturated suspension and then 0.1 to 20 volumes of anRBC-isolating solution added. One of skill in the art is aware ofsolutions for isolating RBCs, e.g. lymphocyte or beads with RBC-specificantibodies.

As but an exemplary embodiment, after trituration is completed add HBSScan be added to the cells, then 1 volume of Lymphocyte can be to thebottom of the tube to create a good bilayer. In some embodiments, mixingof the two solutions should be avoided. This mixture can be centrifugedat 1000 g for 10 min Rotate the tube 180° and recentrifuge at 1000 g foran additional 10 min. This will cause the erythrocytes to form a pelletat the bottom of the tube. Using a standard 9″ glass pipette aspiratethe cell suspensions layer between HBSS and Lympholyte is removed andplaced in a new 50 ml tube. HSBB can be added to the suspension to atotal volume of 20 ml of HBSS and then the suspension mixed by pipettingvia a 5 ml pipette for 1 minutes.

In a next step, the cells are isolated from the HBSS solution. As butone example, the cells can be pelleted by centrifugation in a centrifugetube for from about 800 rpm to about 1600 rpm for from about 1 minute toabout 20 minutes. As but one example, the cells can be pelleted bycentrifugation in a centrifuge tube for about 1200 rpm for 5 minutes. Insome embodiments, the In a next step, the cells can be resuspended inHBSS, with the resulting suspension having a pH of from about 5.0 toabout 6.0. In some embodiments, the resulting suspension can have a pHof from about 5.4 to about 5.8. In some embodiments, the resultingsuspension can have a pH of from about 5.6 to about 5.7. In someembodiments, the resulting suspension can have a pH of about 5.6. Insome embodiments, the HBSS solution prior to admixture with the cellscan have a pH of from about 5.0 to about 5.7. In some embodiments, theHBSS solution prior to admixture with the cells can have a pH of fromabout 5.3 to about 5.6. In some embodiments, the HBSS solution prior toadmixture with the cells can have a pH of about 5.4. In someembodiments, the cells can be resuspended at a concentration of fromabout 2×10⁴ cells/mL to about 2×10⁸ cells/mL. In some embodiments, thecells can be resuspended at a concentration of about 2×10⁶.

As but an exemplary example the resuspension step of the preceedingparagraph can be performed as follows: when making the solution acidic,mildly pipette it using a 5 ml pipette for 10 seconds immediately afteradding the acid to the Hanks Solution. HBSS has a very weak bufferingcapacity, so any solution transferred from the supernatant of theprevious suspension will affect the pH of the HBSS drastically. Theinstructions below will show how to create HBSS with the optimum pH of5.6-5.7 for STAP cell generation according to this experimentalembodiment. First, titrate the pH of pre-chilled HBSS (at 4 degrees C.)with 12N HCl to a pH of 5.6. This is done by slowly adding 11.6 ul of 12N HCl to 50 ml of HBSS. After confirming this pH, sterilize the solutionby filtering through a 0.2 micron syringe filter or bottle top filterof, into a new sterile container for storage. Please confirm the finalpH of 5.6-5.7 through an initial test experiment with an appropriatenumber of cells. Because the pH of the HBSS is so important, the pH ofthe solution be checked, re-titrated and re-sterilized prior to eachuse.

In a next step, the cells in the HBSS suspension can be incubated atabout their in vivo temperature. For example, mammalian cells can beincubated at about 37° C. In some embodiments, the incubation can be forfrom about 5 minutes to about 3 hours. In some embodiments, theincubation can be for from about 10 minutes to about 1 hour. In someembodiments, the incubation can be for from about 15 minutes to about 40minutes. In some embodiments, the incubation can be for about 25minutes.

In a next step, the cells are isolated from the acidic HBSS solution. Asbut one example, the cells can be pelleted by centrifugation in acentrifuge tube for from about 800 rpm to about 1600 rpm for from about1 minute to about 20 minutes. As but one example, the cells can bepelleted by centrifugation in a centrifuge tube for about 1200 rpm for 5minutes. In some embodiments, the supernatant can then be aspirated.

In a next step, the cells can be resuspended in media suitable formaintaining and/or selecting a pluripotent cell. In some embodiments,the media is sphere media. As used herein, “sphere media refers toDMEM/F12 with 1% Antibiotic and 2% B27 Gibco 12587-010. In someembodiments, the media can further comprise growth factors, e.g., b-FGF(20 ng/ml), EGF (20 ng/ml), heparin (0.2%, Stem Cell Technologies07980). In some embodiments, LIF (1000 U) can be added if the cells aremurine). In some embodiments, supplements such as bFGF, EGF and heparinmay not be necessary.) In some embodiments, the cells can be resuspendedin media at a concentration of 10⁵ cells/cc.

In a next step, the cells can be cultured and/or maintained, e.g. at 37°C. with 5% CO₂. In some embodiments, the cells can be agitated duringculturing/maintaining to prevent adherence to a cell culture container.In some embodiments, the cells can be gently pipetted using a 5 mlpipette, twice/day for 2 minutes, for the first week, to discourage themfrom attaching to the bottom of the dishes. In some embodiments, thiscan promote good sphere formation. In some embodiments, sphere media,optionally containing supplements, can be added every other day. Forexample, add 1 ml/day to a 10 cm culture dish, or 0.5 ml/day to a 6 cmdish.

In one aspect, described herein is a method of treating neurologicaldamage in a vertebrate, the method comprising administering to thevertebrate pluripotent (including “more pluripotent” cells as describedherein) cells or STAP cells as described herein to a vertebrate in needof treatment for neurological damage. In some embodiments the cellsadministered are cells generated by the improved methods describedherein, e.g. the methods of the two immediately foregoing aspects and/orExample 5. In some embodiments, the cells can be administered in ascaffold, hydrogel, or delayed-release formulation. In some embodiments,the cells can be autologous to the vertebrate. In some embodiments, thecells are generated from neurological tissue. In some embodiments, thevertebrate is in need of treatment for neurotoxin exposure, acuteneurological injury, chronic neurological injury, and/or a degenerativeneurological disease. In some embodiments, the neurological damage cancomprise damage to the spinal cord, nerves, and/or brain. hi someembodiments, the vertebrate can be a rodent, e.g. a mouse or rat. Insome embodiments, the vertebrate can be a canine, a feline, a dog, acat, a domesticated animal, a horse, or a primate, e.g. a human. In someembodiments, the method can comprise repeated administrations, e.g. 2 ormore, 3 or more, 4 or more or more administrations. In some embodiment,the cells can be administered to the site of the damage, e.g. surgicallyimplanted and/or injected.

In one aspect, provided herein is a kit comprising a pipette having anopening of from about 90 to about 200 microns in diameter and/or apipette having an opening of from about 25 microns to about 90 micronsin diameter. In some embodiments the first pipette has an opening offrom about 100 to about 150 microns in diameter and the second pipettehas an opening of from about 50 microns to about 70 microns in diameter.

In some embodiments, the kit can further comprise an additional pipettehaving an opening of from about 0.5 mm to about 2.0 mm in diameter. Insome embodiments, the pipette can have an opening of from about 0.7 mmto about 1.5 mm in diameter. hi some embodiments, the pipette can havean opening of about 1.1 mm diameter.

In some embodiments, kits can alternatively be provided with deviceshaving apertures and/or lumens of the diameters described above forpipettes, e.g. microfluidics devices having channels with apertures orlumens with the described internal diameters.

In some embodiments, the kit can further comprise HBSS. In someembodiments, the HBSS can have a pH of from about 5.0 to about 5.7. Insome embodiments, the HBSS can have a pH of from about 5.3 to about 5.6.In some embodiments, the HBSS can have a pH of about 5.4. In someembodiments, the kit can further comprise acid for titrating the pH ofthe HBSS. In some embodiments, the acid is HCl. In some embodiments, thekit can further comprise sphere media, and optionally, growth factors.

A kit is any manufacture (e.g., a package or container) comprising atleast one multi-electrode array according to the various embodimentsherein, the manufacture being promoted, distributed, or sold as a unitfor performing the methods or assays described herein. The kitsdescribed herein include reagents and/or components that permit thegeneration, culture and/or selection of pluripotent cells. The kitsdescribed herein can optionally comprise additional components usefulfor performing the methods and assays described herein. Such reagentscan include, e.g. cell culture media, growth factors, differentiationfactors, buffer solutions, labels, imaging reagents, and the like. Suchingredients are known to the person skilled in the art and may varydepending on the particular cells and methods or assay to be carriedout. Additionally, the kit may comprise an instruction leaflet and/ormay provide information as to the relevance of the obtained results.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. These and other changes can be made to the disclosure inlight of the detailed description.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A method to generate a pluripotent cell, comprising        subjecting a cell to a stress.    -   2. The method according to paragraph 1, wherein the pluripotent        cell is generated without introduction of an exogenous gene, a        transcript, a protein, a nuclear component or cytoplasm, or        without cell fusion.    -   3. The method of any of paragraphs 1-2, further comprising        selecting a cell exhibiting pluripotency.    -   4. The method of any of paragraphs 1-3, wherein the cell is not        present as part of a tissue.    -   5. The method of any of paragraphs 1-4, wherein the cell is a        somatic cell, a stem cell, a progenitor cell or an embryonic        cell.    -   6. The method of any of paragraphs 1-5, wherein the cell is an        isolated cell.    -   7. The method of any of paragraphs 1-6, wherein the cell is        present in a heterogeneous population of cells.    -   8. The method of any of paragraphs 1-7, wherein the cell is        present in a homogenous population of cells.    -   9. The method of any of paragraphs 1-8, wherein selecting the        cell exhibiting pluripotency comprises selecting a cell        expressing a stem cell marker.    -   10. The method of any of paragraph 9, wherein the stem cell        marker is selected from the group consisting of:        -   Oct4; Nanog; E-cadherin, and SSEA4.    -   11. The method of any of paragraphs 1-10, wherein selecting the        cell exhibiting pluripotency comprises selecting a cell which is        not adherent.    -   12. The method of any of paragraphs 1-11, wherein the stress        comprises unphysiological stress in tissue or cell culture.    -   13. The method of any of paragraphs 1-12, wherein the stress        comprises exposure of the cell to at least one environmental        stimulus selected from: trauma, mechanical stimuli, chemical        exposure, ultrasonic stimulation, oxygen-deprivation, radiation,        exposure to extreme temperatures, dissociation, trituration,        physical stress, hyperosmosis, hypoosmosis, membrane damage,        toxin, extreme ion concentration, active oxygen, IJV exposure,        strong visible light, deprivation of essential nutrition, or        unphysiolosically acidic environment.    -   14. The method of any of paragraphs 1-13, wherein the stress        comprises exposing the cell to a pH of from about 3.0 to about        6.8.    -   15. The method of any of paragraphs 1-14, wherein the stress        comprises exposing the cell to a pH of from about 4.5 to about        6.0.    -   16. The method of paragraph 15, wherein the stress comprises        exposing the cell to a pH of from about 5.4 to about 5.8.    -   17. The method of any of paragraphs 12-16, wherein the cell is        exposed for 2-3 days.    -   18. The method of any of paragraphs 12-17, wherein the cell is        exposed for 1 day or less.    -   19. The method of any of paragraphs 12-18, wherein the cell is        exposed for 1 hour or less.    -   20. The method of any of paragraphs 12-19, wherein the cell is        exposed for about 30 minutes.    -   21. The method of paragraph 13, wherein the exposure to extreme        temperatures comprises exposing the cell to temperatures below        35° C. or above 42° C.    -   22. The method of paragraph 21, wherein the exposure to extreme        temperatures comprises exposing the cell to temperatures at, or        below freezing or exposure of the cell to temperatures at least        about 85° C.    -   23. The method of paragraph 13, wherein the mechanical stimulus        comprises exposing the cell to shear stress or/and high        pressure.    -   24. The method of paragraph 23, wherein the mechanical stimulus        comprises passing the cell through at least one device with a        smaller aperture than the size of the cell.    -   25. The method of paragraph 23, wherein the mechanical stimulus        comprises passing the cell through several devices having        progressively smaller apertures.    -   26. The method of any of paragraphs 1-25, further comprising        culturing the pluripotent cell to allow propagation of the        pluripotent cell.    -   27. The method of any of paragraphs 1-26, wherein the        pluripotent cell expresses a stem cell marker.    -   28. The method of paragraph 27, wherein the stem cell marker is        selected from the group consisting of:        -   Oct4; Nanog; E-cadherin, and SSEA4.    -   29. The method of any of paragraphs 1-28, wherein the cell is a        mammalian cell.    -   30. The method of any of paragraphs 1-29, wherein the cell is a        human cell.    -   31. The method of any of paragraphs 1-30, wherein the cell is an        adult cell, a neonatal cell, a fetal cell, amniotic cell, or        cord blood cell.    -   32. The method of any of paragraphs 1-31, further comprising        maintaining the pluripotent cell in vitro.    -   33. The method of any of paragraphs 1-32, wherein the epigenetic        state of the cell is altered to more closely resemble the        epigenetic state of an embryonic stem cell.    -   34. The method of paragraph 33, wherein the epigenetic state        comprises methylation patterns.    -   35. The method of any of paragraphs 1-34, wherein the stress        comprises removing at least about 40% of the cytoplasm from the        cell.    -   36. The method of paragraph 35, wherein at least about 50% of        the cytoplasm is removed from the cell.    -   37. The method of paragraph 36, wherein at least about 60% of        the cytoplasm is removed from the cell.    -   38. The method of paragraph 37, wherein between 60-80% of the        cytoplasm is removed from the cell.    -   39. The method of paragraph 37, wherein at least about 80% of        the cytoplasm is removed from the cell.    -   40. The method of paragraph 39, wherein at least about 90% of        the cytoplasm is removed from the cell.    -   41. The method of any of paragraphs 1-40, wherein the stress        comprises removing at least about 40% of the mitochondria from        the cell.    -   42. The method of paragraph 41, wherein the removal of a portion        of the cytoplasm removes at least about 50% of the mitochondria        from the cytoplasm.    -   43. The method of paragraph 42, wherein the removal of cytoplasm        or mitochondria removes about 50%-90% of the mitochondria from        the cytoplasm.    -   44. The method of paragraph 42, wherein the removal of cytoplasm        or mitochondria removes more than 90% of the mitochondria from        the cytoplasm.    -   45. The method of any of paragraphs 1-44, wherein the stress is        sufficient to disrupt the cellular membrane of at least 10% of        cells exposed to the stress.    -   46. An assay comprising;        -   contacting a pluripotent cell produced by the method            according to any of paragraphs 1 to 45 with a candidate            agent.    -   47. The assay of paragraph 46, for use to identify agents which        affect one or more of the viability, differentiation,        proliferation of the pluripotent cell.    -   48. Use of a pluripotent cell produced by the method according        to any one of paragraphs 1 to 45 in a method of cell therapy for        a subject.    -   49. A method of preparing a cell or tissue that is compatible        with cell therapy to be administered to a subject, comprising:        -   generating a pluripotent cell from a cell according to any            one of paragraphs 1 to 45;        -   wherein the cell is an autologous cell or HLA-matched            allogeneic cell.    -   50. The method of paragraph 49, further comprising        differentiating the pluripotent cell along a pre-defined cell        lineage prior to administering the cell or tissue to the        subject.    -   51. A composition comprising a pluripotent cell, wherein the        pluripotent cell is generated from a cell by the methods        according any of paragraphs 1 to 45.    -   52. A method of producing a pluripotent stem cell, the method        comprising culturing a cell in the presence of        adrenocorticotropic hormone (ACTH), 2i or 3i medium    -   53. The method of paragraph 52, wherein the cell is cultured in        LIF medium comprising ACTH.    -   54. The method of paragraph 52 or 53, wherein the ACTH is        present at a concentration of from about 0.1 μM to about 100 μM.    -   55. The method of any of paragraphs 52-54, wherein the cell is a        cell generated by the method of any of paragraphs 1-45.    -   56. The method of any of paragraphs 52-55, wherein the cell is a        totipotent cell.    -   57. The method of any of paragraphs 52-56, wherein the cell is        cultured in the presence of ACTH, 2i or 3i medium for at least 3        days.    -   58. The method of any of paragraphs 52-57, wherein the cell is        cultured in the presence of ACTH, 2i or 3i medium for at least 5        days.    -   59. The method of any of paragraphs 52-58, wherein the cell is        cultured in the presence of ACTH, 21 or 3i medium for at least 7        days.    -   60. The method of any of paragraphs 52-59, wherein after the        culturing step, the cell expresses detectable level of a stem        cell marker selected from the group consisting of: Oct3/4;        Nanog; Rex1; Klf4; Sox2; Klf2; Esrr-beta; Tbx3; and Klf5.    -   61. A method of increasing the self-renewal ability of a        pluripotent cell, the method comprising culturing the cell in        the presence of adrenocorticotropic hormone (ACTH), 2i or 3i        medium.    -   62. The method of paragraph 61, wherein the cell is cultured in        LIF medium comprising ACTH.    -   63. The method of any of paragraphs 61-62, wherein the ACTH is        present at a concentration of from about 0.1 μM to about 100 μM.    -   64. The method of any of paragraphs 61-63, wherein the cell is a        cell generated by the method of any of paragraphs 1-45.    -   65. The method of any of paragraphs 61-64, wherein the cell is a        totipotent cell.    -   66. The method of any of paragraphs 61-65, wherein the cell is        cultured in the presence of ACTH, 2i or 3i medium for at least 3        days.    -   67. The method of any of paragraphs 61-66, wherein the cell is        cultured in the presence of ACTH, 2i or 3i medium for at least 5        days.    -   68. The method of any of paragraphs 61-67, wherein the cell is        cultured in the presence of ACTH, 2i or 3i medium for at least 7        days.    -   69. The method of any of paragraphs 61-68, wherein after the        culturing step, the cell expresses detectable level of a stem        cell marker selected from the group consisting of: Oct3/4;        Nanog; Rex1; Klf4; Sox2; Klf2; Esrr-beta; Tbx3; and Klf5.    -   70. A method of autologous cell therapy in a subject in need of        cell therapy, comprising        -   a. generating a pluripotent cell from a cell according to            any one of paragraphs 1 to 45, wherein the cell is obtained            from the subject, and        -   b. administering a composition comprising the pluripotent            cell or a differentiated progeny thereof to the subject.    -   71. The method of paragraph 70, further comprising        differentiating the pluripotent cell along a pre-defined cell        lineage prior to administering the composition to the subject.    -   72. A method of producing a pluripotent cell capable of        differentiating into a placental cell, the method comprising        culturing the pluripotent cell generated by the method of any of        paragraphs 1-45 in the presence of FGF4.    -   73. The method of paragraph 72, wherein the concentration of        FGF4 is 1 nM to 1 uM.    -   74. The method of paragraph 72 or 73, wherein the pluripotent        cell is capable of differentiating into an embryonic stem cell.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method to generate a pluripotent cell, comprising:    -   a. Isolating an initial cell from a solution;    -   b. Resuspending a cell resulting from step a in Hanks Balanced        Saline Solution (HBSS);    -   c. Triturating the cell suspension resulting from step b;    -   d. Adding from about 2 to about 20 volumes of HBSS to the cell        suspension;    -   e. Isolating a cell from the suspension resulting from step d;    -   f. Resuspending the cell resulting from step e in HBSS having a        pH of about 5.0 to about 6.0;    -   g. Incubating the cells at about their natural in vivo        temperature;    -   h. Isolating a cell from the suspension resulting from step g;        and    -   i. Resuspending the cell pellet resulting from step h in media.-   2. The method of paragraph 1, wherein isolating comprises    centrifugation.-   3. The method of any of paragraphs 1-2, further comprising    contacting the initial cell with trypsin for about 1 minute to about    10 minutes prior to step a.-   4. The method of paragraph 3, further comprising contacting the    initial cell with trypsin for about 3 minutes to about 5 minutes    prior to step a.-   5. The method of any of paragraphs 3-4, wherein the trypsin is    deactivating by contacting the cell pellet with Dulbecco's Minimal    Essential Medium (DMEM)/F-12, comprising 10% heat-inactivated fetal    bovine serum (FBS).-   6. The method of any of paragraphs, 1-5, wherein the trituation of    step c comprises triturating the cells through a series of apertures    or lumens of progressively smaller diameters.-   7. The method of paragraph 6, wherein the series comprises at least    3 apertures or lumens.-   8. The method of any of paragraphs 6-7, wherein at least the first    aperture or lumen is pre-coated with HBSS or water.-   9. The method of any of paragraphs 6-8, wherein the first aperture    or lumen has an internal diameter of from about 0.5 mm to about 2.0    mm.-   10. The method of paragraph 9, wherein the first aperture or lumen    has an internal diameter of from about 0.7 mm to about 1.5 mm.-   11. The method of paragraph 10, wherein the first aperture or lumen    has an internal diameter of about 1.1 mm.-   12. The method of any of paragraphs 6-11, wherein the trituration    through the first aperture or lumen is performed for from about 1    minute to about 10 minutes.-   13. The method of paragraph 12, wherein the trituration through the    first aperture or lumen is performed for about 5 minutes.-   14. The method of any of paragraphs 6-13, wherein the last two    apertures or lumens in the series have internal diameters of from    about 90 to about 200 microns and from about 25 microns to about 90    microns.-   15. The method of paragraph 14, wherein the last two apertures or    lumens in the series have internal diameters of from about 100 to    about 150 microns and from about 50 microns to about 70 microns.-   16. The method of any of paragraphs 1-15, wherein the trituration    comprises about 5 to about 20 minutes of trituration through the    second to last aperture or lumen and about 5 to about 20 minutes of    trituration in the last aperture or lumen.-   17. The method of paragraph 16, wherein the trituration comprises    about 10 minutes of trituration through the second to last aperture    or lumen and about 15 minutes of trituration in the last aperture or    lumen.-   18. The method of any of paragraphs 6-17, wherein the trituration in    the last aperture or lumen is continued until the suspension passes    easily through the aperture or lumen.-   19. The method of any of paragraphs 6-18, wherein the trituration in    each aperture or lumen is continued until the suspension passes    easily through that aperture or lumen.-   20. The method of any of paragraphs 1-19, wherein the total time of    trituation is about 30 minutes.-   21. The method of any of paragraphs 1-20; wherein about 5 to about    15 volumes of HBSS are added in step d.-   22. The method of any of paragraphs 1-21; wherein about 10 volumes    of HBSS are added in step d.-   23. The method of any of paragraphs 1-22, wherein the pH of the HBSS    of step f is from about 5.1 to about 5.7.-   24. The method of any of paragraphs 1-23, wherein the pH of the HBSS    of step f is about 5.4.-   25. The method of any of paragraphs 1-24, wherein the pH of the cell    suspension in HBSS resulting from step f is from about 5.0 to about    6.0.-   26. The method of any of paragraphs 1-25, wherein the pH of the cell    suspension in HBSS resulting from step f is from about 5.6 to about    5.7.-   27. The method of any of paragraphs 1-26, wherein step f comprises    resuspending the cells to a concentration of from about 0.5 million    cells/mL to about 4 million cells/mL.-   28. The method of any of paragraphs 1-27, wherein step f comprises    resuspending the cells to a concentration of about 2 million    cells/mL.-   29. The method of any of paragraphs 1-28, wherein step g comprises    incubating the cells about about 37° C. for about 25 minutes.-   30. The method of any of paragraphs 1-29, wherein steps g and h    combined last from about 10 minutes to about 1 hour.-   31. The method of any of paragraphs 1-30, wherein steps g and h    combined last for about 30 minutes.-   32. The method of any of paragraphs 1-31, wherein the media of step    i is Sphere Media comprising DMEM/F12, about 1% antibiotic, about 2%    B27, and optionally, one or more growth factors.-   33. The method of paragraph 32, wherein the growth factors comprises    bFGF, EGF, and heparin.-   34. A method to generate a pluripotent cell, wherein an initial cell    is present in a tissue comprising red blood cells, the method    comprising:    -   a. Mechanically slicing the tissue in the presence of one or        more ECM-degrading enzymes;    -   b. Incubating the sample resulting from step a at about the        tissue's natural in vivo temperature while agitating the tissue;    -   c. Diluting the cell suspension resulting from step bin HBSS;    -   d. Isolating a cell from the suspension resulting from step c;    -   e. Resuspending a cell resulting from step d in HBSS;    -   f. Triturating the cell suspension resulting from step e;    -   g. Adding from about 0.1 to about 10 volumes of an RBC-isolating        solution the cell suspension resulting from step f to create a        bilayer;    -   h. Separating the HBSS layer resulting from step g from the        RBC-isolating solution layer;    -   i. Isolating a cell from the HBSS suspension resulting from step        h;    -   j. Resuspending the cell resulting from step i in HBSS with a pH        of about 5.0 to about 6.0;    -   k. Incubating the cells at about the tissue's natural in vivo        temperature;    -   l. Isolating a cell from the suspension resulting from step k;        and    -   m. Resuspending the cell resulting from step 1 in media.-   35. The method of paragraph 34, wherein the tissue comprising red    blood cells is selected from the group consisting of:    -   lung; spleen; and liver.-   36. The method of any of paragraphs 34-35, wherein the tissue is    lung and the ECM-degrading enzyme is collagenase P.-   37. The method of any of paragraphs 34-36, wherein the slicing of    step a is continued for about 10 minutes.-   38. The method of any of paragraphs 34-37, wherein the trituation of    step f comprises triturating the cells through a series of apertures    or lumens of progressively smaller diameters.-   39. The method of paragraph 38, wherein the series comprises at    least 3 apertures or lumens.-   40. The method of any of paragraphs 38-39, wherein at least the    first aperture or lumen is pre-coated with HBSS or water.-   41. The method of any of paragraphs 38-40, wherein the first    aperture or lumen has an internal diameter of from about 0.5 mm to    about 2.0 mm.-   42. The method of paragraph 41, wherein the first aperture or lumen    has an internal diameter of from about 0.7 mm to about 1.5 mm.-   43. The method of paragraph 42, wherein the first aperture or lumen    has an internal diameter of about 1.1 mm.-   44. The method of any of paragraphs 38-43, wherein the trituration    through the first aperture or lumen is performed for from about 1    minute to about 10 minutes.-   45. The method of paragraph 44, wherein the trituration through the    first aperture or lumen is performed for about 5 minutes.-   46. The method of any of paragraphs 38-45, wherein the last two    apertures or lumens in the series have internal diameters of from    about 90 to about 200 microns and from about 25 microns to about 90    microns.-   47. The method of paragraph 46, wherein the last two apertures or    lumens in the series have internal diameters of from about 100 to    about 150 microns and from about 50 microns to about 70 microns.-   48. The method of any of paragraphs 38-47, wherein the trituration    comprises about 5 to about 20 minutes of trituration through the    second to last aperture or lumen and about 5 to about 20 minutes of    trituration in the last aperture or lumen.-   49. The method of paragraph 48, wherein the trituration comprises    about 10 minutes of trituration through the second to last aperture    or lumen and about 15 minutes of trituration in the last aperture or    lumen.-   50. The method of any of paragraphs 34-49, wherein the trituration    in the last aperture or lumen is continued until the suspension    passes easily through the aperture or lumen.-   51. The method of any of paragraphs 34-50, wherein the trituration    in each aperture or lumen is continued until the suspension passes    easily through that aperture or lumen.-   52. The method of any of paragraphs 34-51, wherein the total time of    trituation is about 30 minutes.-   53. The method of any of paragraphs 34-52, wherein the pH of the    HBSS of step j is from about 5.1 to about 5.7.-   54. The method of paragraph 53, wherein the pH of the HBSS of step j    is about 5.4.-   55. The method of any of paragraphs 34-54, wherein the pH of the    cell suspension in HBSS resulting from step j is from about 5.0 to    about 6.0.-   56. The method of paragraph 55, wherein the pH of the cell    suspension in HBSS resulting from step j is from about 5.6 to about    5.7.-   57. The method of any of paragraphs 34-56, wherein step k comprises    incubating the cells about about 37° C. for about 25 minutes.-   58. The method of any of paragraphs 34-57, wherein steps k and l    combined last from about 10 minutes to about 1 hour.-   59. The method of any of paragraphs 34-58, wherein the media of step    m is Sphere Media comprising DMEM/F12, 1% antibiotic, 1% B27, and    optionally, one or more growth factors.-   60. The method of paragraph 59, wherein the growth factors comprises    bFGF, EGF, and heparin.-   61. The method of any of paragraphs 1-60, wherein the initial cell    is a murine cell and the Sphere Media comprises LIF.-   62. The method of any of paragraphs 1-61, further comprising a step    of culturing the resulting cells for at least one week, the    culturing comprising:    -   a. Adding sphere media, optionally comprising growth factors;    -   b. Agitating the cells to discourage attachment to the bottom of        the dish.-   63. The method of paragraph 62, wherein the sphere media is added    every 1-4 days.-   64. The method of paragraph 63, wherein the sphere media is added    every 2 days.-   65. The method of any of paragraphs 62-64, wherein the agitation    comprises pipetting the cells with a pipette-   66. The method of paragraph 65, wherein the pipette has a opening of    about 1.1 mm in diameter.-   67. The method of any of paragraphs 65-66, wherein the pipetting is    performed at least once per day.-   68. The method of paragraph 67, wherein the pipetting is performed    at least twice per day.-   69. The method of any of paragraphs 1-68, further comprising    selecting a cell with pluripotency, wherein the selecting comprises    a method selected from the group consisting of:    -   selecting cells with low adherency; selecting cells that are a        component of a sphere; and    -   selecting cells with a small relative size.-   70. The method of any of paragraphs 1-69, further comprising a final    step of selecting a cell exhibiting pluripotency.-   71. The method of any of paragraphs 1-70, wherein the initial cell    is not present as part of a tissue.-   72. The method of any of paragraphs 1-71, wherein the initial cell    is a somatic cell, a stem cell, a progenitor cell or an embryonic    cell.-   73. The method of any of paragraphs 1-72, wherein the initial cell    is an isolated cell.-   74. The method of any of paragraphs 1-73, wherein the initial cell    is present in a heterogeneous population of cells.-   75. The method of any of paragraphs 1-74, wherein the initial cell    is present in a homogenous population of cells.-   76. The method of any of paragraphs 1-75, wherein selecting the cell    exhibiting pluripotency comprises selecting a cell expressing a stem    cell marker.-   77. The method of any of paragraph 76, wherein the stem cell marker    is selected from the group consisting of:    -   Oct4; Nanog; E-cadherin, and SSEA4.-   78. The method of any of paragraphs 1-77, wherein selecting the cell    exhibiting pluripotency comprises selecting a cell which is not    adherent.-   79. The method of any of paragraphs 1-78, further comprising    culturing the pluripotent cell to allow propagation of the    pluripotent cell.-   80. The method of any of paragraphs 1-79, wherein the pluripotent    cell expresses a stem cell marker.-   81. The method of paragraph 80, wherein the stem cell marker is    selected from the group consisting of:    -   Oct4; Nanog; E-cadherin, and SSEA4.-   82. The method of any of paragraphs 1-81, wherein the initial cell    is a mammalian cell.-   83. The method of any of paragraphs 1-82, wherein the initial cell    is a human cell.-   84. The method of any of paragraphs 1-83, wherein the initial cell    is an adult cell, a neonatal cell, a fetal cell, amniotic cell, or    cord blood cell.-   85. The method of any of paragraphs 1-84, further comprising    maintaining the pluripotent cell in vitro.-   86. An assay comprising;    -   contacting a pluripotent cell produced by the method according        to any of paragraphs 1 to 85 with a candidate agent.-   87. The assay of paragraph 86, for use to identify agents which    affect one or more of the viability, differentiation, proliferation    of the pluripotent cell.-   88. Use of a pluripotent cell produced by the method according to    any one of paragraphs 1 to 85 in a method of cell therapy for a    subject.-   89. A method of preparing a cell or tissue that is compatible with    cell therapy to be administered to a subject, comprising:    -   generating a pluripotent cell from a cell according to any one        of paragraphs 1 to 85;    -   wherein the cell is an autologous cell or HLA-matched allogeneic        cell.-   90. The method of paragraph 89, further comprising differentiating    the pluripotent cell along a pre-defined cell lineage prior to    administering the cell or tissue to the subject.-   91. A composition comprising a pluripotent cell, wherein the    pluripotent cell is generated from a cell by the methods according    any of paragraphs 1 to 85.-   92. A method of autologous cell therapy in a subject in need of cell    therapy, comprising    -   a. generating a pluripotent cell from a cell according to anyone        of paragraphs 1 to 85, wherein the cell is obtained from the        subject, and    -   b. administering a composition comprising the pluripotent cell        or a differentiated progeny thereof to the subject.-   93. The method of paragraph 92, further comprising differentiating    the pluripotent cell along a pre-defined cell lineage prior to    administering the composition to the subject.-   94. A kit comprising two pipettes, the first pipette having an    opening of from about 90 to about 200 microns in diameter and the    second pipette having an opening of from about 25 microns to about    90 microns in diameter.-   95. The kit of paragraph 94, wherein the first pipette has an    opening of from about 100 to about 150 microns in diameter and the    second pipette having an opening of from about 50 microns to about    70 microns in diameter.-   96. The kit of any of paragraphs 94-95, further comprising HBSS.-   97. The kit of paragraph 94-96, wherein the HBSS has a pH of about    5.4.-   98. The kit of any of paragraphs 96-97, further comprising acid for    titrating the pH of the HBSS.-   99. The kit of paragraph 98, wherein the acid is HCl.-   100. The kit of any of paragraphs 94-99, further comprising sphere    media, and optionally, growth factors.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method to generate a pluripotent cell, comprising:    -   a. Isolating an initial cell from a solution;    -   b. Resuspending a cell resulting from step a in a solution of        Hanks Balanced Saline Solution (HBSS) and ATP having a pH of        from about 4.0 to about 6.5;    -   c. Triturating the cell suspension resulting from step b;    -   d. Resuspending the cell resulting from step c in HBSS;    -   e. Isolating a cell from the suspension resulting from step d;        and    -   f. Resuspending the cell pellet resulting from step g in media-   2. A method to generate a pluripotent cell, comprising:-   a. Isolating an initial cell from a solution;-   b. Resuspending a cell resulting from step a in a solution of Hanks    Balanced Saline Solution (HBSS) and ATP having a pH of from about    4.0 to about 6.5;-   c. Isolating a cell from the suspension resulting from step b;-   d. Resuspending the cell resulting from step c in a solution of HBSS    and ATP having a pH of from about 4.0 to about 6.5 to the cell    suspension;-   e. Triturating the cell suspension resulting from step d;-   f. Resuspending the cell resulting from step e in HBSS;-   g. Isolating a cell from the suspension resulting from step f; and-   h. Resuspending the cell pellet resulting from step g in media.-   3. The method of any of claims 1-2, wherein the ATP is present in    the solution of HBSS and ATP at a concentration of from about 1.5 to    about 5 mg/cc.-   4. The method of claim 3, wherein the ATP is present in the solution    of HBSS and ATP at a concentration of from about 2.7 mM to about 9    mM.-   5. The method of any of claims 1-4, wherein the solution of HBSS and    ATP having a pH of from about 4.0 to about 6.5 is prepared by    titrating a HBSS solution with a solution of ATP until the desired    pH is achieved.-   6. The method of claim 5, wherein the solution of ATP has a    concentration of from about 50 mM to about 500 mM.-   7. The method of claim 6, wherein the solution of ATP has a    concentration of about 200 mM.-   8. The method of any of claims 1-7, wherein the solution of HBSS and    ATP has a pH of from about 5.0 to about 5.7.-   9. The method of any of claims 1-8, wherein the solution of HBSS and    ATP has a pH of about 5.0.-   10. The method of any of claims 1-9, wherein the HBSS of step f of    claim 62 and step d of claim 61 does not comprise ATP.-   11. The method of any of claims 1-10, wherein isolating comprises    centrifugation.-   12. The method of any of claims 1-11, further comprising contacting    the initial cell with trypsin for about 1 minute to about 10 minutes    prior to step a.-   13. The method of claim 12, further comprising contacting the    initial cell with trypsin for about 3 minutes to about 5 minutes    prior to step a.-   14. The method of any of claims 12-13, wherein the trypsin is    deactivating by contacting the cell pellet with Dulbecco's Minimal    Essential Medium (DMEM)/F-12, comprising 10% heat-inactivated fetal    bovine serum (FBS).-   15. The method of any of claims 1-14, wherein the trituation    comprises triturating the cells through a series of apertures or    lumens of progressively smaller diameters.-   16. The method of claim 15, wherein the series comprises at least 3    apertures or lumens.-   17. The method of any of claims 15-16, wherein at least the first    aperture or lumen is pre-coated with HBSS or water.-   18. The method of any of claims 15-17, wherein the first aperture or    lumen has an internal diameter of from about 0.5 mm to about 2.0 mm.-   19. The method of claim 18, wherein the first aperture or lumen has    an internal diameter of from about 0.7 mm to about 1.5 mm.-   20. The method of claim 19, wherein the first aperture or lumen has    an internal diameter of about 1.1 mm.-   21. The method of any of claims 15-20, wherein the trituration    through the first aperture or lumen is performed for from about 1    minute to about 10 minutes.-   22. The method of claim 21, wherein the trituration through the    first aperture or lumen is performed for about 5 minutes.-   23. The method of any of claims 15-22, wherein the last two    apertures or lumens in the series have internal diameters of from    about 90 to about 200 microns and from about 25 microns to about 90    microns.-   24. The method of claim 23, wherein the last two apertures or lumens    in the series have internal diameters of from about 100 to about 150    microns and from about 50 microns to about 70 microns.-   25. The method of any of claims 1-24, wherein the trituration    comprises about 5 to about 20 minutes of trituration through the    second to last aperture or lumen and about 5 to about 20 minutes of    trituration in the last aperture or lumen.-   26. The method of claim 25, wherein the trituration comprises about    10 minutes of trituration through the second to last aperture or    lumen and about 15 minutes of trituration in the last aperture or    lumen.-   27. The method of any of claims 15-26, wherein the trituration in    the last aperture or lumen is continued until the suspension passes    easily through the aperture or lumen.-   28. The method of any of claims 15-27, wherein the trituration in    each aperture or lumen is continued until the suspension passes    easily through that aperture or lumen.-   29. The method of any of claims 1-28, wherein the total time of    trituation is about 30 minutes.-   30. The method of any of claims 1-29; wherein about 5 to about 15    volumes of HBSS after the trituration.-   31. The method of any of claims 1-30; wherein about 10 volumes of    HBSS after the trituration.-   32. The method of any of claims 1-31, wherein the pH of the HBSS    added after trituration is from about 5.1 to about 5.7.-   33. The method of any of claims 1-32, wherein the pH of the HBSS    added after trituration is about 5.4.-   34. The method of any of claims 1-33, wherein the pH of the cell    suspension in HBSS resulting from step f of claim 62 or step d of    claim 61 is from about 5.0 to about 6.0.-   35. The method of any of claims 1-33, wherein the pH of the cell    suspension in HBSS resulting from step f of claim 62 or step d of    claim 61 is from about 5.6 to about 5.7.-   36. The method of any of claims 1-35, wherein after the addition of    HBSS after trituration, the cells are present at a concentration of    from about 0.5 million cells/mL to about 4 million cells/mL.-   37. The method of any of claims 1-36, wherein after the addition of    HBSS after trituration, the cells are present at a concentration of    about 2 million cells/mL.-   38. The method of any of claims 1-37, wherein the media is Sphere    Media comprising DMEM/F12, about 1% antibiotic, about 2% B27, and    optionally, one or more growth factors.-   39. The method of claim 38, wherein the growth factors comprises    bFGF, EGF, and heparin.-   40. A method to generate a pluripotent cell, wherein an initial cell    is present in a tissue comprising red blood cells, the method    comprising:-   a. Mechanically slicing the tissue in the presence of one or more    ECM-degrading enzymes;-   b. Incubating the sample resulting from step a at about the tissue's    natural in vivo temperature while agitating the tissue;-   c. Diluting the cell suspension resulting from step b in a solution    of Hanks Balanced Saline Solution (HBSS) and ATP having a pH of from    about 4.0 to about 6.5;-   d. Triturating the cell suspension resulting from step c;-   e. Resuspending the cell resulting from step d in HBSS;-   f. Isolating a cell from the suspension resulting from step e; and-   g. Resuspending the cell pellet resulting from step g in media.-   41. A method to generate a pluripotent cell, wherein an initial cell    is present in a tissue comprising red blood cells, the method    comprising:-   a. Mechanically slicing the tissue in the presence of one or more    ECM-degrading enzymes;-   b. Incubating the sample resulting from step a at about the tissue's    natural in vivo temperature while agitating the tissue;-   c. Diluting the cell suspension resulting from step b in a solution    of Hanks Balanced Saline Solution (HBSS) and ATP having a pH of from    about 4.0 to about 6.5;-   d. Isolating a cell from the suspension resulting from step b;-   e. Resuspending the cell resulting from step c in a solution of HBSS    and ATP having a pH of from about 4.0 to about 6.5 to the cell    suspension;-   f. Triturating the cell suspension resulting from step d;-   g. Resuspending the cell resulting from step e in HBSS;-   h. Isolating a cell from the suspension resulting from step f; and-   i. Resuspending the cell pellet resulting from step g in media.-   42. The method of any of claims 40-41, wherein the tissue comprising    red blood cells is selected from the group consisting of:    -   lung; spleen; and liver.-   43. The method of any of claims 40-42, wherein the tissue is lung    and the ECM-degrading enzyme is collagenase P.-   44. The method of any of claims 40-43, wherein the slicing of step a    is continued for about 10 minutes.-   45. The method of any of claims 40-44, wherein the ATP is present in    the solution of HBSS and ATP at a concentration of from about 1.5 to    about 5 mg/cc.-   46. The method of claim 45, wherein the ATP is present in the    solution of HBSS and ATP at a concentration of from about 2.7 mM to    about 9 mM.-   47. The method of any of claims 40-46, wherein the solution of HBSS    and ATP having a pH of from about 4.0 to about 6.5 is prepared by    titrating a HBSS solution with a solution of ATP until the desired    pH is achieved.-   48. The method of claim 47, wherein the solution of ATP has a    concentration of from about 50 mM to about 500 mM.-   49. The method of claim 48, wherein the solution of ATP has a    concentration of about 200 mM.-   50. The method of any of claims 40-49, wherein the solution of HBSS    and ATP has a pH of from about 5.0 to about 5.7.-   51. The method of any of claims 40-50, wherein the solution of HBSS    and ATP has a pH of about 5.0.-   52. The method of any of claims 40-50, wherein the HBSS of step g of    claim 101 and step e of claim 100 does not comprise ATP.-   53. The method of any of claims 40-50, wherein isolating comprises    centrifugation.-   54. The method of any of claims 40-53, further comprising contacting    the initial cell with trypsin for about 1 minute to about 10 minutes    prior to step c.-   55. The method of claim 54, further comprising contacting the    initial cell with trypsin for about 3 minutes to about 5 minutes    prior to step c.-   56. The method of any of claims 54-55, wherein the trypsin is    deactivating by contacting the cell pellet with Dulbecco's Minimal    Essential Medium (DMEM)/F-12, comprising 10% heat-inactivated fetal    bovine serum (FBS).-   57. The method of any of claims 40-57, wherein the trituation    comprises triturating the cells through a series of apertures or    lumens of progressively smaller diameters.-   58. The method of claim 57, wherein the series comprises at least 3    apertures or lumens.-   59. The method of any of claims 40-58, wherein at least the first    aperture or lumen is pre-coated with HBSS or water.-   60. The method of any of claims 40-59, wherein the first aperture or    lumen has an internal diameter of from about 0.5 mm to about 2.0 mm.-   61. The method of claim 60, wherein the first aperture or lumen has    an internal diameter of from about 0.7 mm to about 1.5 mm.-   62. The method of claim 61, wherein the first aperture or lumen has    an internal diameter of about 1.1 mm.-   63. The method of any of claims 40-62, wherein the trituration    through the first aperture or lumen is performed for from about 1    minute to about 10 minutes.-   64. The method of claim 40-63, wherein the trituration through the    first aperture or lumen is performed for about 5 minutes.-   65. The method of any of claims 40-64, wherein the last two    apertures or lumens in the series have internal diameters of from    about 90 to about 200 microns and from about 25 microns to about 90    microns.-   66. The method of claim 65, wherein the last two apertures or lumens    in the series have internal diameters of from about 100 to about 150    microns and from about 50 microns to about 70 microns.-   67. The method of any of claims 40-66, wherein the trituration    comprises about 5 to about 20 minutes of trituration through the    second to last aperture or lumen and about 5 to about 20 minutes of    trituration in the last aperture or lumen.-   68. The method of claim 67, wherein the trituration comprises about    10 minutes of trituration through the second to last aperture or    lumen and about 15 minutes of trituration in the last aperture or    lumen.-   69. The method of any of claims 40-68, wherein the trituration in    the last aperture or lumen is continued until the suspension passes    easily through the aperture or lumen.-   70. The method of any of claims 40-69, wherein the trituration in    each aperture or lumen is continued until the suspension passes    easily through that aperture or lumen.-   71. The method of any of claims 40-70, wherein the total time of    trituation is about 30 minutes.-   72. The method of any of claims 40-71; wherein about 5 to about 15    volumes of HBSS after the trituration.-   73. The method of any of claims 40-72; wherein about 10 volumes of    HBSS after the trituration.-   74. The method of any of claims 40-73, wherein the pH of the HBSS    added after trituration is from about 5.1 to about 5.7.-   75. The method of any of claims 40-74, wherein the pH of the HBSS    added after trituration is about 5.4.-   76. The method of any of claims 40-75, wherein the pH of the cell    suspension in HBSS resulting from step g of claim 101 or step e of    claim 100 is from about 5.0 to about 6.0.-   77. The method of any of claims 40-76, wherein the pH of the cell    suspension in HBSS resulting from step g of claim 101 or step e of    claim 100 is from about 5.6 to about 5.7.-   78. The method of any of claims 40-77, wherein after the addition of    HBSS after trituration, the cells are present at a concentration of    from about 0.5 million cells/mL to about 4 million cells/mL.-   79. The method of any of claims 40-78, wherein after the addition of    HBSS after trituration, the cells are present at a concentration of    about 2 million cells/mL.-   80. The method of any of claims 40-79, wherein the media is Sphere    Media comprising DMEM/F12, about 1% antibiotic, about 2% B27, and    optionally, one or more growth factors.-   81. The method of claim 80, wherein the growth factors comprises    bFGF, EGF, and heparin.-   82. The method of any of claims 1-81, wherein the initial cell is a    murine cell and the Sphere Media comprises LIF.-   83. The method of any of claims 1-81, further comprising a step of    culturing the resulting cells for at least one week, the culturing    comprising:-   j. Adding sphere media, optionally comprising growth factors;-   k. Agitating the cells to discourage attachment to the bottom of the    dish.-   84. The method of claim 83, wherein the sphere media is added every    1-4 days.-   85. The method of claim 83, wherein the sphere media is added every    2 days.-   86. The method of any of claims 82-85, wherein the agitation    comprises pipetting the cells with a pipette-   87. The method of claim 86, wherein the pipette has a opening of    about 1.1 mm in diameter.-   88. The method of any of claims 86-87, wherein the pipetting is    performed at least once per day.-   89. The method of claim 88, wherein the pipetting is performed at    least twice per day.-   90. The method of any of claims 1-89, further comprising selecting a    cell with pluripotency, wherein the selecting comprises a method    selected from the group consisting of:    -   selecting cells with low adherency; selecting cells that are a        component of a sphere; and selecting cells with a small relative        size.-   91. The method of any of claims 1-90, further comprising a final    step of selecting a cell exhibiting pluripotency.-   92. The method of any of claims 1-91, wherein the initial cell is    not present as part of a tissue.-   93. The method of any of claims 1-92, wherein the initial cell is a    somatic cell, a stem cell, a progenitor cell or an embryonic cell.-   94. The method of any of claims 1-93, wherein the initial cell is an    isolated cell.-   95. The method of any of claims 1-94, wherein the initial cell is    present in a heterogeneous population of cells.-   96. The method of any of claims 1-95, wherein the initial cell is    present in a homogenous population of cells.-   97. The method of any of claims 1-96, wherein selecting the cell    exhibiting pluripotency comprises selecting a cell expressing a stem    cell marker.-   98. The method of any of claim 97, wherein the stem cell marker is    selected from the group consisting of:    -   Oct4; Nanog; E-cadherin, and SSEA4.-   99. The method of any of claims 1-98, wherein selecting the cell    exhibiting pluripotency comprises selecting a cell which is not    adherent.-   100. The method of any of claims 1-99, further comprising culturing    the pluripotent cell to allow propagation of the pluripotent cell.-   101. The method of any of claims 1-100, wherein the pluripotent cell    expresses a stem cell marker.-   102. The method of claim 101, wherein the stem cell marker is    selected from the group consisting of:    -   Oct4; Nanog; E-cadherin, and SSEA4.-   103. The method of any of claims 1-102, wherein the initial cell is    a mammalian cell.-   104. The method of any of claims 1-103, wherein the initial cell is    a human cell.-   105. The method of any of claims 1-104, wherein the initial cell is    an adult cell, a neonatal cell, a fetal cell, amniotic cell, or cord    blood cell.-   106. The method of any of claims 1-105, further comprising    maintaining the pluripotent cell in vitro.-   107. An assay comprising;    -   contacting a pluripotent cell produced by the method according        to any of claims 1 to 106 with a candidate agent.-   108. The assay of claim 107, for use to identify agents which affect    one or more of the viability, differentiation, proliferation of the    pluripotent cell.-   109. Use of a pluripotent cell produced by the method according to    any one of claims 1 to 106 in a method of cell therapy for a    subject.-   110. A method of preparing a cell or tissue that is compatible with    cell therapy to be administered to a subject, comprising:    -   generating a pluripotent cell from a cell according to any one        of claims 1 to 106;    -   wherein the cell is an autologous cell or HLA-matched allogeneic        cell.-   111. The method of claim 110, further comprising differentiating the    pluripotent cell along a pre-defined cell lineage prior to    administering the cell or tissue to the subject.-   112. A composition comprising a pluripotent cell, wherein the    pluripotent cell is generated from a cell by the methods according    any of claims 1 to 106.-   113. A method of autologous cell therapy in a subject in need of    cell therapy, comprising-   c. generating a pluripotent cell from a cell according to any one of    claims 1 to 106, wherein the cell is obtained from the subject, and-   d. administering a composition comprising the pluripotent cell or a    differentiated progeny thereof to the subject.-   114. The method of claim 113, further comprising differentiating the    pluripotent cell along a pre-defined cell lineage prior to    administering the composition to the subject.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method of treating neurological damage, the method comprising    administering a pluripotent or STAP cell to a subject in need of    treatment for neurological damage.-   2. The method of claim 1, wherein the pluripotent or STAP cell is    generated by the methods described herein, e.g. of paragraphs [0157]    to [0190], the numbered paragraphs of paragraphs [00202]-[00204],    Example 5, or Example 7.-   3. The method of any of claims 1-2, wherein the cell is autologous    to the subject.-   4. The method of any of claims 1-3, wherein the neurological damage    is selected from the group consisting of:    -   acute neurological damage; chronic neurological damage;        degenerative neurological disease; nerve injury; or spinal cord        injury.

EXAMPLES Example 1

All organisms possess a primitive survival instinct. When plants aresubjected to significant external stresses they activate a mechanism tosurvive that causes dedifferentiation of cells and enables regenerationof the injured area or the entire organism. While such mechanisms appearto be essential for lower organisms to survive extreme environmentalchanges, they have yet to be documented in mammals.

The inventors hypothesized that physical stress may cause maturemammalian cells to revert to a stem cell state, similar to that seen inplants and lower organisms. To examine this hypothesis, mature cellsprocured from seven adult somatic tissues were studied. To first focuson which physical stresses might be most effective in altering maturecells to revert to a less mature state, CD45 positive lymphocytesharvested from Oct4-GFP mice were studied. Cells from these mice providea readout of reversion to a stem cell phenotype when the stem cellspecific Oct4 promoter is activated. The mature, fully differentiatedcells were exposed to several significant external stimuli.

For example, CD45 positive lymphocytes were exposed to low pH solutionto provide a strong chemical stress. Within 3 days of exposure, GFPexpressing cells were observed, and within 5 days, spherical coloniescomposed of GFP expressing cells were observed. Cells generated in thismanner are referred to in this Example as Stress Altered Stem Cells(SASCs or SACs). SACs can also be referred to as rejuvenated stem cells(RSCs) or animal callus cells (ACCs). SACs expressed several markersnormally associated with embryonic stem cells. SACs exhibited adifferentiation potency equivalent to ES cells, contributed to thegeneration of chimera mice and were capable of generating whole fetuseswhen injected into 4N blastocysts. Cells generated in this mannerinitially showed low mitochondrial activity and other conditionsnormally associated with the induction of cell based injury defensemechanisms. They then exhibited demethylation of the Oct4 and Nanog genepromoters. The reprogramming of stress altered cells appeared to beinduced via mesenchymal-epithelial transition. The findings areconsistent with descriptions of cells contained in the plant callus, inresponse to injury (external stimuli). A plant callus is formed from astress induced conversion of cells to pluripotent plant stem cells,capable of forming clonal bodies. Such a spherical colony, generatedfrom mature fully differentiated somatic mammalian cells in response tosignificant external stimuli, is referred to herein as an Animal Callus,and to the stress altered cells contained in such a colony or callus, as“Animal Callus Cells” (ACCs) or SACs.

Thus, significant physical and chemical stresses caused normal matureadult cells to be reprogrammed to pluripotent stem cells capable ofembryogenesis. While not wishing to be bound by theory, the mechanism ofreprogramming appears to include the induction of a cellular survivaland repair process normally seen in response to injury. It isdemonstrated herein that mammalian cells possess a survival mechanismvery similar to that of plants, to revert to reprogrammed state inresponse to significant stressful external stimuli.

Various types of cells have reportedly been reprogrammed to apluripotent stem cell state through induction or forced expression ofspecific genes¹⁻⁵. It is also believed that damage to cells as a resultof exposure to irritants, such as burns, chemical injury, trauma andradiation, may alter normal cells to become cancer cells.

Introduction

All organisms appear to have a common instinct to survive injury relatedto stressful stimuli by adapting themselves to the environment andregenerating their bodies. In plants, ontogenesis is observed not onlyin zygotes but also in fully differentiated cells and immature pollen.In vertebrates, newts are capable of regenerating several anatomicalstructures and organs, including their limbs¹. Of particular note isthat the remarkable regenerative capacity demonstrated by both plantsand newts is induced by external stimuli, which cause cellulardedifferentiation of previously fully differentiated somatic cells.While billions of years have passed from the earliest form of life, anddifferent organisms have evolved in unique ways, this survival instinctmay be inherited from a common ancestor to modern-era organisms.Although terminally differentiated mammalian cells are normally believedto be incapable of reversing the differentiation process, mammals mayretain a previously unappreciated program to escape death in response todrastic environmental changes.

The plant callus, a mass of proliferating cells formed in response toexternal stimuli, such as wounding, which can be stimulated in cultureby the plant hormones². The callus contains reprogrammed somatic cells,referred to as callus cells, each of which is capable of clonallyregenerating the entire body. Callus cells are not inherent in plants,but are generated from somatic cells in response to external stimuli.Although recent studies demonstrated that mammalian somatic cells can bereprogrammed by exogenous processes, such as gene induction³⁻⁷,reprogramming of mammalian somatic cells in response to externalphysical and or chemical stimuli, in a manner that parallels plants, hasnot been reported. Interestingly, it is believed that extreme externalstimuli, such as exposure to irritants, including burns, chemicalinjury, trauma and radiation, may alter normal somatic cells to becomecancer cells. Such experiences seem to indicate that external stimuliwill result in mammalian cellular change.

In this study, it was hypothesized that mammalian cells retain amechanism to survive exposure to significant external stress, in thesame manner as plants. This report presents evidence that application ofsignificant physical and chemical stimuli can cause reprogramming ofmature, fully differentiated mammalian somatic cells, procured fromvarious tissues, and that such stress altered cells are capable offorming an animal callus containing “animal callus cells”, which canregenerate the clonal body.

Results

Significant Physical and Chemical Stimuli Applied to Mature SomaticCells.

Since the embryonic transcription factor Oct4 is thought to be crucialin regulation of the pluripotent status of cells, the initial strategywas to identify which external stimuli most efficiently altered maturecells to become reprogrammed to express Oct4. CD45 positivehematopoietic lineage cells were first studied in order to avoidcontamination with undifferentiated cells. CD45 positive cells harvestedfrom spleens procured from Oct4-GFP (GOF) mice⁸, were exposed to varioussignificant physical and chemical stimuli. The exposures included:osmotic pressure treatment, treatment with significant mechanicaltrituration, exposure to low pH, application of cell membrane damageusing streptolysin O (SLO), exposure to under nutrition and exposure tohypoxia and high Ca²⁺ concentration. Next, GFP expressing cells wereidentified, sorted and collected using FACS. Gene expression of Oct4 wasconfirmed by R-T PCR. Exposure to each of the applied stimuli resultedin reprogramming of the mature cells to express GFP to some degree (FIG.5A). Exposure of the mature cells to the chemical stress of low pH andthe physical stress of significant mechanical trituration appeared to bethe most effective treatments in altering mature cells to express Oct4.To determine the optimal pH for inducing conversion to Oct4 expressingcells, CD45 positive cells were exposed to solutions of varying acidity,from pH 4.0 to pH 6.8. At 3 days after exposure to an acidic solution,GFP expression of cells was analyzed using FACS. An acid solution with apH 5.4-5.6 most efficiently altered cells to express GFP (FIG. 5B).Consequently, exposure to low pH was focused upon as the stresstreatment of choice for the remainder of the study.

The optimum culture conditions for maintaining stress altered Oct4expressing cells were then determined Several previously describedculture media, including: ES establishment culture medium, 3i⁹ andACTH¹⁰, ES culture condition, ES-LIF¹¹, embryonic neural stem cellculture condition, B27-LIF¹², and EpiSCs culture condition¹³, werestudied. Cells were plated into each medium, and GFP expressed colonieswere counted (FIG. 5C). The medium B27-LIF appeared to be the mosteffective in generating GFP expressing spherical colonies. ThereforeB27-LIF medium was utilized for culture of the treated cells.

Stress treated CD45 positive cells were cultured in B27-LIF medium, andwithin 5 days, GFP expressing spherical colonies were observed while noGFP expressing colonies were observed in the untreated control (FIG.1A). Spherical colonies grew to approximately 70 μm in diameter over thefirst 7 days, and spherical colonies could be maintained for another 7days in that culture condition. The configuration of the colonies wasslightly baroque, appearing more similar in shape to the callus seen inbotany, rather than spheres. A cell colony generated by stress treatmentwas therefore referred to as an Animal Callus (AC). Cultured cells weredissociated and population analysis was then performed using FACS. Theanalysis revealed that the application of certain significant stimuliresulted in the generation of stress altered cells, now referred to asAnimal Callus Cells (ACCs), that did not previously exist in the CD45positive cell populations (FIG. 1B). The phenotypic change of CD45positive cells as a result stress treatment was observed at the singlecell level. While CD45 positive cells did not express GFP, ACCsexpressed GFP associated with a diminished expression of CD45 (data notshown). Examination of single cells revealed that the cell size oftreated cells appeared smaller than untreated cells. Therefore, cellsize of ACCs population was analyzed by FACS. The cell size of ACCs wasquite small, with 80% of cells being less than 8 μm in diameter (FIG.1C).

To examine chronological phenotypic change associated with CD45diminution and Oct4 expression, stress treated CD45 positive cells wereanalyzed at day 1, day 3 and day 7. At day 1, most of cells stillexpressed CD45, but not Oct4. At day 3, marker expression transitionedto reveal CD45 negative cells or CD45 negative/Oct4positive (dim) cells.At day 7, CD45 expression disappeared, and Oct4 expressing cells wereobserved (FIG. 1D). Notably, during the first 7 days of culture, thenumber of PI positive cells (dead cells) gradually increased (Data notshown), which suggested that the stress treatment and the culturecondition gradually changed the character of cells and selected forsuccessfully altered cells, which expressed Oct4.

Characterization of ACCs.

To confirm the reprogramming of somatic cells as a result of exposure toexternal stimuli, early embryogenesis marker gene expression of ACCs wasinvestigated. As a positive control of early embryogenesis, ES cellswere utilized in following experiments. Marker expression and DNAmethylation was characterized as follows: Immunofluorescence staining atday 7, showed that spherical colonies containing ACCs, uniformlyexpressed pluripotent cell markers, E-cadherin antigen, Nanog, S SEA-1,PCAM-1, and AP, and were positive for Oct4-GFP (data not shown). Geneexpression analysis showed that ACCs and ES cells, but not primary CD45positive cells, expressed comparable levels of Oct4, Nanog, Sox2, Ecat1,Esg1, Dax1, Fgf5, Klf4 and Rex1 genes (FIG. 2A). Gene expression of ESspecific genes in ACCs reached a peak at day 7 (FIG. 2A). Bisulfitesequencing was performed to determine the methylation status of Oct4 andNanog gene promoters in ACCs. Native lymphocytes and cultured lymphocytecontrol samples displayed extensive methylation at both promoters,whereas ACCs showed widespread demethylation of these regions similar tothat seen in ES cells (FIG. 2B). Thus, it is demonstrated that mammaliansomatic cells were reprogrammed by external stress.

To confirm that the Oct4 gene expression resulted from stress treatmentof mature cells not only in GOF mice but also in wild type mice, CD45positive lymphocytes were harvested from spleens procured from ICR mice.The lymphocytes were then exposed to the stress treatment andchronologically analyzed until day 7 using FACS. A SSEA-1positive/E-cadherin positive cell population was seen in the stresstreated group, while SSEA-1/E-cadherin expression was not observed inthe non-stress treated control group (FIG. 6A). Those double positivecells expressed Oct4 gene expression, which was confirmed by R-T PCR(FIG. 6B). These results demonstrated that as a result of the stresstreatment, ACCs, Oct4 positive and pluripotent marker expressing cells,were generated from CD45 positive cells irrespective of mouse strain.

These results imply that the mature fully differentiated adult somaticcells reverted to “stemness” as a result of the stress treatment.

To assess the stemness of ACCs, their self-renewal potency and theirdifferentiation potency were examined. To study their self-renewalpotency, ACCs colonies derived from previously mature CD45 positivelymphocytes were dissociated into single cells, and plated into 96 wellplates, with one cell per well in an effort to generate clonally derivedpopulations. Ten days after plating, spherical colonies were seen in 4of the 96 wells. The dividing time of ACCs varied from well to well.Some divided in 12-16 h and others divided in 30-34 h. ACCs werepassaged at least 5 times, with continued expression of Oct4 observed.Consequently, ACCs demonstrated a potential for self-renewal, and thepotential to differentiate into cells from all three germ layers invitro.

ACs derived from mature GOF lymphocytes were again dissociated intosingle cells, sorted to contain only a population of cells thatexpressed GFP and then cultured in differentiation media. At 14-21 daysafter plating, cells expressed the ectoderm marker, βIII-tubulin andGFAP, the mesoderm marker, α-smooth muscle actin, and the endodermmarker, α-fetoprotein and Cytokeratin 7 (data not shown). Thus, ACCsdifferentiated into cells representative of the three germ layers invitro.

Stress Alteration of Mature Somatic Cells Procuredfrom Various AdultTissues.

To examine whether ACCs could be generated not only mature lymphocytesbut also other types of somatic cells, brain, skin, muscle, fat, bonemarrow, lung and liver were harvested from Oct4-GFP (GOF) mice⁸. Cellswere isolated from the tissue samples, dissociated into single cells,and treated with different physical and or chemical stress conditions.The efficiency of the process to alter the cells differed as a functionof both the source of cells and the stress condition(s) to which thecells were exposed (FIG. 7A). The ability of stress to alter maturecells to express Oct4, differed depending on the derivation of cells,but stress was able to alter cells to express Oct4 to some degree inmature cells derived from all three germ layers (FIG. 7A). ACC coloniesderived from any mature tissue expressed pluripotent markers,E-cadherin, Nanog, PCAM-1 and AP (data not shown), and ES specificmarker genes (FIG. 7B). Significant physical and chemical stressesaltered mature somatic cells to revert to a stem cell state, despite ofthe source of tissues and derivation of the germ layers.

Cellular Modification in the Initial Phase of ACCs Generation.

These results demonstrate that strong physical and chemical stimuliresult in reprogramming of somatic cells. Stress treated lymphocyteswere observed to form an AC within 5 days. It was hypothesized thatdrastic change of molecular events occurred as a result of the stressexposure. Studies were therefore focused on the initial phase of thereprogramming, which was the during the first 7 days after the exposureto the stimuli.

Because ACCs survived after the significant stress exposure, it wasspeculated that survival mechanisms normally turned on to repaircellular damage were induced during the ACCs generation. First theexpression of a number of candidate genes involved in cellular responseto stress and DNA repair¹⁴ was compared in in native CD45 positive cellsand stress-treated CD45 positive cells at day 1, day 3 and day 7.Cellular response gene expression was already observed at day 1, andthose genes were up-regulated over 7 days when the mixtures of ACCgenerating cells and other cells were analyzed (FIG. 8). Because theup-regulation of cellular response genes was correlated with ACCsgeneration, ACCs at day 3 and day 7 were sorted, and gene expression wasanalyzed. With the exception of Hif3a, all candidate genes wereup-regulated to various degrees during the ACCs generation (FIG. 3A).Four heat shock genes and one DNA repair gene were found to beup-regulated during the ACCs generation. Furthermore, seven of theup-regulated genes are known to be directly involved in the regulationof the cellular redox state. These results suggested that theself-repair or self-defense potency was induced during the ACCsgeneration.

Since ACCs exhibited the up-regulation of cellular redox associatedgenes, the mitochondrial function of ACCs was next examined.Mitochondria are organelles responsible for production of the vastmajority of ATP via the redox reaction using oxygen within eukaryoticcells. GFP expression of ACC spherical colonies gradually diminishedfrom peripheral located cells after 7 days when colonies were culturedwithout passage. ACCs contained at day 10 contained GFP expressingcentral cells and non-GFP differentiated peripheral cells (data notshown). Mitochondrial morphology was evaluated in ACCs anddifferentiated cells by staining with a mitochondrial-specific dye,MitoTracker Red. ACC mitochondria were observed as pen-nuclear clustersthat appear punctuate and globular while differentiated cell containedmany mitochondria which were filamentous and wide-spread in cytoplasm.ATP production of ACCs was less than that in native CD45 positive cells(FIG. 3B). Also, reactive oxygen species (ROS) production of ACCs wasless than in native CD45 positive cells (FIG. 3C). Finally the keyfactors involved in mtDNA replication were assessed; which aremitochondrial transcription factor A (Tfam), the mitochondrial-specificDNA polymerase gamma (Polg) and its accessory unit (Polg2). The geneexpression of Tfam, Polg, and Polg2 in ACCs was lower than those indifferentiated cells (FIG. 3D). Consequently, ACCs contained smallnumbers of mitochondria and ACCs' mitochondrial activity was lower thandifferentiated cells. These results implied that ACCs acquired ametabolic system distinct from differentiated cells to survive after thesevere stress response.

Developmental Potential of ACCs.

Finally, it was assessed whether ACCs possessed a developmentalpotential similar to that of plant callus cells. As an initial test fordevelopmental potency, ACCs implanted subcutaneously in immunodeficient(SCID) mice were studied. Six weeks after transplantation, ACCsgenerated tissues representing all three germ layers (data not shown).

ACCs differentiated into cells representative of all three germ layersin vivo and in vitro. Therefore, the chimera contribution potency ofACCs was assessed. ACCs for use in chimera generation studies wereprepared using CD45 positive cells derived from F1 GFP (C57BL/6GFP×DBA/2or 129/SvGFP×C57BL/6GFP) or GOF. Because gene expression analysis hadrevealed that at day 7, ACCs expressed the highest level of pluripotentmarker genes, day 7 ACCs were utilized for the chimera mouse generationstudy. Initially, conventional methods for chimera generation wereutilized. ACs were dissociated into single cells via treatment withtrypsin. The ACCs were then injected into blastocysts (FIG. 4A). Usingthis approach, the chimera contribution of dissociated ACCs was quitelow (Table 1). Therefore ACCs without prior trypsin treatment, whichoften causes cellular damage¹⁵, were injected into blastocysts. ACs werecut into small clusters using a micro-knife under the microscopy. Smallclusters of ACs were then injected into blastocysts (FIG. 4A). Usingthis approach, the chimera contribution of ACCs dramatically increased(data not shown). Chimera mice generated with ACCs grew up healthy (datanot shown) and germ line transmission has been observed. The chimeracontribution rate of each tissue was analyzed by FACS. The resultsshowed that ACCs derived from lymphocytes contributed to all tissue(FIG. 4B).

As demonstrated above, ACCs can be generated from various cells derivedfrom all three germ layers (FIG. 7A-7B). In order to examine whetherACCs derived from various tissues had different differentiationtendencies, ACCs were generated from various tissues derived from F1 GFPmice, and injected into ICR blastocysts. Then, using FACS, thecontribution ratio of each tissue in the generated chimera mice wasanalyzed. It was found that ACCs derived from any tissue contributed tochimeric mouse generation (FIG. 9). In addition, the contribution ratioto skin, brain, muscle, fat, liver and lung was analyzed in chimera micegenerated using ACCs derived from various tissues. ACCs derived from anytissue contributed to generate tissues representative of all three germlayers, and no differentiation tendency was observed (FIG. 9).

The generation of mice by tetraploid complementation, which involvesinjection of pluripotent cells in 4N host blastocysts, represents themost rigorous test for developmental potency because the resultingembryos are derived only from injected donor cells¹⁶ ACCs were generatedfrom lymphocytes derived from DBA×B6GFP F1 mice or 129/SvGFP×B6GFP F1.ACCs resulted in the generation of (mid) late-gastration ‘all ACCembryos’ after injection into 4N blastocysts (data not shown).Genotyping analysis demonstrated that ‘all ACC embryos’ had specificgenes of strain which was utilized to generate ACCs. Thus, ACCspossessed the potential to generate a clonal body just like plant calluscells.

Discussion

Mammalian somatic cells exhibit the ability for animal callus (AC)formation as a result of exposure to significant external stimuli, in afashion very similar to plants. The cells contained in these calli(animal callus cells, ACCs) have the ability to generate chimeric miceand to generate new embryos fully consisting of only cells generatedfrom ACCs. The results described herein demonstrate that mammaliansomatic cells regain the ability to differentiate into any of the threegerm layers by external stimuli. This implies that somatic cells have agreater plasticity than previously believed. Furthermore, this studydemonstrates the potential of somatic cell reprogramming without geneinduction or the introduction of foreign proteins, and offers newinsight into the potential of adult stem cells; representing asignificant milestone in the elucidation of stem cell biology.

Materials and Methods

Tissue Harvesting and Cell Culture.

For mature lymphocytes isolation, spleens derived from GOF mice or ICRmice were minced by scissors and mechanically-dissociated with pasturepipettes. Dissociated spleens were strain through a cell strainer (BDBiosciences, San Jose). Collected cells were re-suspended in DMEM mediumand added the same volume of lympholyte (CEDARLANE®, Ontario, Canada),then centrifuged at 1000 g for 15 min Lymphocytes layer was taken outand attained with CD45 antibody (ab25603, abcam, Cambridge, Mass.). CD45positive cells were sorted by FACS Aria (BD Biosciences). Then, CD45positive cells were treated with stress treatment (pH5.5 solution for 15min) and plated into B27 medium supplemented with 1000 U LIF (Sigma) and10 ng/ml FGF 2 (Sigma).

Exposure to External Stimuli—Stress Treatment.

To give a mechanical stress to mature cells, pasture pipette were heatedand then stretched to create lumens approximately 50 microns indiameters, and then broken. Mature somatic cells were then trituratedthrough these pipettes for 20 min, and cultured for 7 days. To provide ahypoxic stimulus to mature cells, cells were cultured in a 5% oxygenincubator for 3 weeks. An under nutrition stimulus was provided tomature cells, by culturing the cells in a basic culture medium for 3weeks. To expose the mature cells to a physiological stress, they weretreated with low pH (pH5.5) solution, and cultured for 7 days. Also,cells were given more serious damage. To create pores in mature cellmembranes, cells were treated with SLO (Streptolysin O). SLO-treatedcells were incubated in HBSS containing 10 μg/mL SLO at 37° C. for 50min and then cultured in culture medium without SLO for 7 days. Cellsexposed to under-nutrition stress were cultured in basal medium for 2 to3 weeks. Cells exposed to “ATP” stress were incubated in HBSS containing2.4 mM ATP at 37° C. for 15 min and then cultured in culture medium for7 days. Cells exposed to “Ca” stress were cultured in culture mediumcontaining 2 mM CaCl₂ for 2 weeks.

Bisulfate Sequence.

For cells procured from GOF mice were dissociated into single cells. GFPpositive cells collected using by FACS Aria. Genome DNA was extractedfrom ACCs and studied. Bisulfate treatment of DNA was done using theCpGenome DNA Modification Kit (Chemicon, Temecula, Calif.,http://www.chemicon.com) following the manufacturer's instructions. Theresulting modified DNA was amplified by nested polymerase chain reactionPCR using two forward (F) primers and one reverse (R) primer: Oct4 (F1,GTTGTTTTGTTTTGGTTTTGGATAT (SEQ ID NO: 1; F2, ATGGGTTGAAATATTGGGTTTATTTA(SEQ ID NO: 2); R,CCACCCTCTAACCTTAACCTCTAAC (SEQ ID NO: 3)). And Nanog(F1, GAGGATGTTTTTTAAGTTTTTTTT (SEQ ID NO:4); F2,AATGTTTATGGTGGATTTTGTAGGT (SEQ ID NO: 5); R, CCCACACTCATATCAATATAATAAC(SEQ ID NO:6)). PCR was done using TaKaRa Ex Taq Hot Start Version(RR030A). DNA sequencing was performed using M13 primer with theassistance of GRAS (The Genome Resource and Analysis Unit).

Immunohistochemistry.

Cultured cells were fixed with 4% paraformaldehyde and permeabilizedwith 0.1% Triton X-100/PBS prior blocking with 1% BSA solution (LifeTechnology, Tokyo, Japan). Secondary antibodies were goat anti-mouse or-rabbit coupled to Alexa-488 or -594 (Invitrogen). Cell nuclei werevisualized with DAPI (Sigma). Slides were mounted with SlowFade Goldantifade reagent (Invitrogen).

Fluorescence-Activated Cell Sorting and Flow Cytometry.

Cells were prepared according to standard protocols and suspended in0.1% BSA/PBS on ice prior to FACS. PI (BD Biosciences) was used toexclude dead cells. Cells were sorted on a BD FACSAria SORP and analyzedon a BD LSRII with BD FACSDiva Software (BD Biosciences).

RNA Preparation and RT-PCR Analysis.

RNA was isolated with the RNeasy Micro kit (QIAGEN). Reversetranscription was performed with the SupeSACript III First StrandSynthesis kit (Invitrogen). SYBR Green Mix I (Roche Diagnostics) wasused for amplification, and samples were run on a Lightcycler-IIInstrument (Roche Diagnostics).

Animal Studies.

For tumorigenicity studies, cells suspended in 100 ml PBS were injectedsubcutaneously in the flanks of age-matched immunodeficient SCID mice.Mice were sacrificed and necropsied after 6 weeks.

ATP and ROS Assay.

Intercellular ATP level was measured by the ATP Bioluminescence AssayKit HS II (Roche) according to supplier's protocol. The luminescenceintensity was measured by using a Gelomax 96 Microplate Luminometer(Promega, Madison, Wis.) and the luminescence readings were normalizedby cell count. For measurement of ROS levels, cells were incubated in amedium contain 2 μM dihydroethidium (Molecular Probes) at 37° C. in darkfor 15 minutes. Cells were then washed with PBS and suspended in PBScontaining 0.5% BSA. The fluorescence intensity of 30000 cells wasrecorded with the help of a BD Biosciences LSR II (BD Bioscience, Spark,Md.).

Chimera Mice Generation and Analyses.

Production of Diploid and Tetraploid Chimeras. Diploid embryos wereobtained from ICR strain females mated with ICR males and tetraploidembryos were obtained from BDF1 strain females mated with BDF1 males.Tetraploid embryos were produced by the electrofusion of 2-cellembryos¹⁷. In this study, because trypsin treatment caused lowchimerism, ACCs spherical colonies were cut into small pieces using amicro-knife under the microscopy, then small clusters of ACCs wereinjected into day 4.5 blastocyst by large pipette. Next day, thechimeric blastocysts were transferred into day 2.5 pseudopregnantfemales.

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TABLE 1 Generation of chimera mice from ACCs Cell No. of No. of chimericpreparation Culture fertilized mice obtained Mouse for period of embryosHigh strain injection SACs injected No. offspring Total contribution**BDF1 Single 7 day 40 32 1 0 BDF1 Cluster 7 day 58  48* 16 4 129B6F1Cluster 7 day 98 64 20 6 GOF Cluster 7 day 73 35 24 2 GOF Cluster 10day  35 20 4 0 *All fetuses were collected at 13.5 dpc to 15.5 dpc andthe contribution rate of ACCs into each organs was examined by FACS**The contribution of SACs into each chimera was scored as high (>50% ofthe coat color of GFP expression)

TABLE 2Primer sequences. The middle column contains, from top to bottom, SEQID NOs: 7-39 and the right hand column contains, from top to bottom,SEQ ID NOs: 40-72. Gene 5′ Primer 3′ Primer Txni gtcatccttgatctgcccctgagacgacctgctacacctg Bmi1 ggtacttacgatgcccagca tccctacctgactgcttacgPrdx2 ccctgaatatccctctgct tatgtctgctcgtacccctt Hspb1agatggctacatctctcggt tcagacctcggttcatcttc Hif3a cactctggacttggagatgccttggaccttcgaaggacga Hspa1b cttgtcgttggtgatggtga tcaaagcgcagaccacctcgHspa9a gttgaagcagttaatatggc gcatgtcgtccgcagtaact Ercc4agatgagaccaacctggacc tcgacttcgtcttgttcggt Hpas1a aggtggagatcatcgccaactctacctgttccgcgtctag Gapdh cgttgaatttgccgtgagtg tggtgaaggtcggtgtgaacGpx2 attgccaagtcgttctacga gtaggacagaaacggatgga Sod2 aggtcgcttacagattgctggtgtcgatcgttcttcact Tgr gtctctttagaaaagtgtga attgcagctgcaaatccctg Gstatacagctttcttcctggcca tacgattcacggaccgtgcc Pdha2 atgtcagccttgtggaaattaacgataactgatccctggg Gpx3 gtctgacagaccaataccat cagttctacctgtaggacag Gpx4aacggctgcgtggtgaagcg cctccttccaggtctccgga Polg ggacctcccttagagagggaagcatgccagccagagtcact Pol2 acagtgccttcaggttagtc actccaatctgagcaagaccTfam gcatacaaagaagctgtgag gttatatgctgaacgaggtc Oct4tctttccaccaggcccccggct tgcgggcggacatggggagatcc Ecat1tgtggggccctgaaaggcgagctgagat atgggccgccatacgacgacgctcaact Esg1gaagtctggttccttggcaggatg actcgatacactggcctagc Nanog caggtgtttgagggtagctccggttcatcatggtacagtc ERas actgcccctcatcagactgctactcactgccttgtactcgggtagctg Gdf3 qttccaacctqtqcctcqcqtcttaqcqaqqcatqqaqaqaqcqqaqcaq Fgf4 cgtggtgagcatcttcggagtggccttcttggtccgcccgttctta Rex1 acgagtggcagtttcttcttgggatatgactcacttccagggggcact Cripto atggacgcaactgtgaacatgatgttcgcactttgaggtcctggtccatcacgtgaccat Dax1 tgctgcggtccaggccatcaagaggggcactgttcagttcagcggatc Sox2 tagagctagactccgggcgatgattgccttaaacaagaccacgaaa Klf4 gcgaactcacacaggcgagaaacctcgcttcctcttcctccgacaca Fgf5 gctgtgtctcaggggattgt cactctcggcctgtcttttc

TABLE 3 Percent of cells demonstrating pluripotent phenotype after 1week of stress treatment. Treatments are shown in the first column andthe tissue of origin of the somatic cells is shown in the second row.Numbers are percentages. 1 week-old Bone Fibro- Marrow Brain Lung MuscleFat blast Control 0 0 0 0 0 0 Hypoxia 2 3 3.2 2.8 1.6 1.2 Trituration19.5 20.5 19.8 20.6 18.4 9.5 SLO 13.2 10.3 18.4 20.5 32.8 15.2undernutrition 2 3.4 1.8 4.5 2.4 1.5 ATP 12.3 15.4 9.8 68.4 79.6 25.10Ca 1.2 0.8 1.3 1.5 2.7 3.5

Example 2 Stimulus-Triggered Fate Conversion of Somatic Cells intoPluripotency

Described herein is a phenomenon for nuclear initialization,‘stimulus-triggered acquired pluripotency’ (STAP), where strong externalstimuli sufficiently reprogram mammalian somatic cells into pluripotentcells, without using nuclear transfer or introducing transcriptionfactors. In the presence of LIF, a transient low-pH stress causesde-differentiation of CD45⁺ hematopoietic cells into cells that expresspluripotent cell markers such as Oct3/4 and have the competence ofthree-germ-layer differentiation. In these STAP cells, like ES cells,substantial demethylation is seen in the oct3/4 and nanog promoterregions. Hematopoietic cell-derived STAP cells carry gene rearrangementsin T cell receptor, indicating that committed somatic cells give rise toSTAP cells by lineage conversion. Blastocyst injection shows that STAPcells efficiently contribute to chimera, even in the tetraploidcomplementation assay, and to offspring via germ-line transmission.Thus, the epigenetic state of fate determination can be radicallyinitialized in a context-dependent manner by strong environmental cues.

In the canalization review of Waddington's epigenetic landscape, fatesof somatic cells are progressively determined as cellulardifferentiation goes downhill. It is generally believed that reversal ofdifferentiated cellular status requires artificial, physical or genetic,manipulation of their nuclear functions such as nuclear transfer¹ andmultiple transcription factor introduction². It remains unansweredwhether somatic cells can undergo initialization of their nuclearprogram simply in response to external triggers without these directnuclear manipulations. Such situations is known to occur in plants;drastic changes in culture environments can convert the fate of maturesomatic cells, e.g., dissociated carrot cells, into immature blastemacells, from which a whole plant structure, including stalks and roots,develops in the presence of auxins. A challenging question is whetheranimal somatic cells may have a similar potential that emerges at leastunder special conditions. Over the last decade, the presence ofpluripotent cells (or closely relevant cell types) in adult tissues hasbeen a matter of debate, for which conflicting conclusions were reportedby various groups. However, none of them have demonstrated that suchpluripotent cells could arise from differentiated somatic cells.

Hematopoietic cells positive for CD45 (leukocyte common antigen) aretypical lineage-committed somatic cells that are often used as startingcell types for reprogramming studies such as derivatization of iPScells. They never express pluripotency-related markers such as Oct3/4unless reprogrammed. In particular, most of CD45⁺ cells from spleentissues are considered to be non-stem leukocyte populations (maturingcells or progenitors), and iPS cell conversion from lymphocytes carryinggenomic rearrangements of T cell receptor 13 chain (tcrβ) gene isregarded as a bona fide hallmark for reprogramming from committedsomatic cells. The inventors therefore became intrigued by the questionwhether splenic CD45⁺ cells may be converted to acquire pluripotency bydrastic changes of external environments such as those caused by simplechemical perturbations.

Results

Low-pH Treatment Induced Fate Conversion in Committed Somatic Cells.

CD45⁺ cells, harvested from adult spleens procured from oct3/4::gfp B6mice¹⁵, were exposed to various types of strong transient stimuli,including physical and chemical ones, and examined for activation of theoct3/4 promoter after culturing in suspension using LIF-containing B27medium for several days. Among these various perturbations, low-pHperturbations were focused on. As shown below, this type ofperturbations turned out to be most effective in oct3/4 induction.

Without exposure to the stimuli, none of cells sorted with CD45expressed oct3/4::GFP regardless of the culture period in LIF-containingmedium, which was permissive for survival of the sorted cells. Incontrast, a 30-minute treatment of splenic CD45⁺ cells with low-pH media(pH4.5-6.0; FIG. 12A) caused the emergence of substantial numbers ofoct3/4::GFP⁺ cells in day-7 (d7) culture (FIG. 12B; the most effectiverange was pH5.4-5.8; FIG. 16B). These cells kept expressing oct3/4::GFPwithout passaging at least for additional 7 days (14 days total). On d7of this non-adhesion culture, low-pH-induced oct3/4::GFP⁺ cells formedspherical (or slightly baroque) clusters (data not shown; consisting ofa few to several dozens cells), which no more expressed CD45 (FIG. 12C).Interestingly, the cell size of low-pH-induced oct3/4::GFP⁺ cells wassubstantially smaller than that of non-treated CD45⁺ cells (seeimmunostaining of oct3/4::GFP and CD45 in a single cell; FIG. 12C); 80%of the former cells were less than 8 μm in diameter while that ofcontrol CD45⁺ cells ranged from 8 to 10 μm (FIG. 12D (left peak showsOct3/4::GFP+ cells and right peak shows CD45+ cells) estimated byforward scattering analysis in FACS). These observations suggestdramatic changes between oct3/4::GFP⁺ and CD45⁺ populations beyond thedifferences in expression of two markers.

The time course analysis (FIG. 12C) showed a dynamic change of cellpopulations during d1-d3. Most of surviving cells on d1 (the survivingcell number corresponded to ˜85% of the d0 population) were still CD45⁺and oct3/4::GFP⁻. On d2 and d3, a substantial population (21% and 34%,respectively) of total surviving cells became oct3/4::GFP⁺ and were dimfor CD45 (FIG. 12C; ˜50-60% of the plated cells were lost by then). Ond7, a significant number of oct3/4::GFP⁺/CD45⁻ cells (54% of totalsurviving cells) constituted a distinct population from theoct3/4::GFP⁻/CD45⁻ one (FIG. 12B, top; total cell numbers on d7 weresimilar to those on d3). No obvious generation of oct3/4::GFP⁺/CD45⁻populations were seen in culture of non-treated CD45⁺ cells (FIG. 12B,bottom). Thus, the number of the oct3/4::GFP⁺/CD45⁻ population in thelow-pH-treated group was fairly substantial and corresponded to about ahalf of the total surviving cells on d7. In fact, when oct3/4::GFPsignals first appeared on d2, the number of GFP⁺ cells corresponded to˜8% of initially plated CD45⁺ cells. Therefore, it appeared unlikelythat a very minor population (e.g., contaminating CD45⁻ cells) quicklygrew to form such a substantial oct3/4::GFP⁺ population over the firsttwo days after the low-pH treatment.

In live imaging analysis (data not shown), low-pH-treated CD45⁺ cells,but not untreated cells, tended to form small clusters, which graduallyturned on GFP signals over the first few days. Then, these smalloct3/4::GFP⁺ clusters frequently fused and formed larger spheres by d5,indicating that the clusters are multi-clonal. Interestingly, these GFP⁺clusters (but not GFP⁻ cells) were quite mobile and often protruded cellprocesses (data not shown).

To test whether lineage-committed splenic CD45⁺ cells, in particular, Tcell populations, contributed to oct3/4::GFP⁺ cells, genomicrearrangements of tcrβ were examined in isolated oct3/4::GFP⁺ spheres bygenomic PCR and it was found that each sphere contained cells with tcrβgene rearrangements (data not shown). To rule out the possibility ofdetecting the rearrangement in contaminating oct3/4::GFP⁻/CD45⁺ cells,oct3/4::GFP⁺/CD45⁻ cells were sorted by FACS on d7 and subjected to thetcrβ gene rearrangement assay. In this case, too, tcrβ generearrangements were clearly observed (FIG. 12E). These findingsdemonstrate that committed somatic cell populations in splenic cells (atleast, T cells) contributed to oct3/4::GFP⁺ cells by converting theirfates from CD45⁺ to oct3/4::GFP⁺.

Low-pH-Induced Oct3/4⁺ Cells have Pluripotency.

It was next examined whether oct3/4::GFP⁺ expression in thestimulus-induced cells represented a pluripotent state of these cells ormerely a specific alteration in the gene expression pattern (in thiscase, oct3/4 and cd45) without acquiring pluripotency. Immunostainingshowed that d7 oct3/4::GFP⁺ spheres expressed pluripotency-relatedmarkers such as Oct3/4, SSEA-1, Nanog, E-cadherin and AP (data notshown). Gene expression analysis by qPCR showed that low-pH-inducedoct3/4::GFP⁺ cells on d7, unlike CD45⁺ cells, expressed comparablelevels of oct3/4, nanog, sox2, ecat1, esg1, dax1 and klf4 genes to thosein ES cells (FIG. 13A (the series represent, from left to right, oct3/4,nanog, sox2, ecat1, esg1, dax1 and klf4 expression); these markers werealready positive on d3), indicating that the low-pH-induced oct3/4::GFP⁺cells express a bona-fide marker gene set characteristic ofpluripotency, which are never expressed in CD45⁺ cells.

It was next tested whether this dramatic alteration in the geneexpression pattern was accompanied by the change in the epigeneticmodification of pluripotency-related genes. To this end, bisulfitesequencing was performed to examine the methylation status of the oct3/4and nanog promoter areas. CD45⁺ cells, with or without additionalculture, displayed heavily methylated patterns at both promoters. Incontrast, low-pH-induced oct3/4::GFP⁺ cells showed extensivedemethylation in these regions, like ES cells (FIG. 13B), demonstratingthat cells underwent a substantial reprogramming of epigenetic status inthese key genes for pluripotency.

It was next examined whether the low-pH-induced cells have a competenceto generate three-germ layer derivatives, which is the common criteriafor the pluripotent nature. Both in vitro differentiation assays (datanot shown) and teratoma-formation test (data not shown) demonstratedthat these cells can give rise to ecodermal (e.g., β-tubulin III⁺),mesodermal (e.g., smooth muscle actin⁺) and endodermal (e.g., alphafetoprotein⁺) cells.

Collectively, these findings demonstrate that the differentiation stateof a committed somatic cell lineage can be converted into a cell stateof pluripotency by strong stimuli given externally. Hereafter, the fateconversion from somatic cells into pluripotent cells by strong externalstimuli such as low pH is referred to as ‘stimulus-triggered acquiredpluripotency’ (STAP) and the resultant cells as STAP cells.

STAP Cells from Other Tissue Sources.

Another important question about STAP cells is whether the phenomenon oflow-pH-triggered conversion is limited to CD45⁺ leukocytes. To addressthis question, similar conversion experiments were performed withsomatic cells harvested from brain, skin, muscle, fat, bone marrow, lungand liver tissues of oct3/4::gfp mice.

Cells from the tissue samples were dissociated into single cells,subjected to a transient low-pH exposure and cultured in LIF-containingmedium. Although conversion efficacy varied among the tissues of theirorigin, oct3/4::GFP⁺ cells were reproducibly observed in d7 culture(FIG. 14A (the series represent, from left to right, CD45+ cells, bonemarrow, brain, lung, muscle, adipose, fibroblasts, liver, andchondrocytes). Notably, STAP cells were efficiently derived frommesechymal cells of adipose tissues (data not shown), where CD45⁺ cellswere rare, and also from primary culture cells of chondrocytes,indicating that non-CD45⁺ cell populations can give rise to STAP cells.These oct3/4::GFP⁺ cell clusters also expressed pluripotency-relatedmarkers (FIG. 14B (the series represent, from left to right, theexpression o Oct3/4, Nanog, Sox2, Klf4, and Rex1) and FIG. 18B data notshown), and ES cell-specific marker genes (FIG. 14B and FIG. 18B).

Characteristics of STAP Cells as Pluripotent Cells.

Thus, STAP cells express ES cell-specific genes and show similarmethylation patterns in oct3/4 and nanog genes. In addition, STAP cellscould be established in culture media for mouse ES cells, such asLIF-containing media, but not in mouse EpiSC medium (data not shown).

However, although STAP cells exhibited a substantial similarity to mouseES cells, several distinct features were also found. For instance, STAPcells showed a limited self-renewal capacity. Unlike mouse ES cells(data not shown), when STAP cell spheres were enzymatically dissociatedinto single cells for clonal culture in each well of a 96-well plate, nocolonies (AP⁺ or oct3/4::GFP⁺) formed after additional 10-day culture inLIF-containing medium (G-MEM- or B27-based) under either adhesive ornon-adhesive conditions (data not shown). Whereas spherical colonyformation was infrequently seen (typically, in 2-4 wells out of the 96wells), these colonies were all AP⁻ and oct3/4::GFP⁻. Even when STAPcell spheres were partially dissociated and cultured under highcell-density conditions (data not shown; presumably more supportive forself-renewal), cell numbers started to decline after two passages andoct3/4::GFP⁺ cells could not be maintained beyond five passages. Thesecharacteristics for growth and maintenance suggest that STAP cellsrepresent a pluripotent cell population whose features are partiallydistinct from mouse ES and iPS cells.

Mouse EpiSCs are another category of pluripotent stem cells, which areconsidered to be slightly more advanced in differentiation stages. STAPcells appeared to behave distinctly from EpiSC cells in several aspects.In adhesion culture, like mouse ES cells, oct3/4::GFP⁺ cells formedhemi-spherical colonies by piling up, unlike mono-layered flat coloniesseen for mouse EpiSCs. STAP cells could not be maintained in EpiSCmedium, either, suggesting that they are dissimilar to EpiSCs (data notshown). In addition, treatment with the ROCK inhibitor, which improvessingle-cell passages of EpiSC (ref; Ohgushi), did not promote colonyformation from dissociated STAP cells (data not shown).

Immunostaining showed that STAP cells were negative for the EpiSCmarkers Claudin 7 and ZO-1 and positive for Klf2/4 (data not shown). Thegrouping between ES cell, STAP cell and EpiSCs might not be so simple,since the expression of the ES cell marker Esrrβ was low in both STAPcells and EpiSCs, while elf5 expression is specifically low in STAPcells (FIG. 15A (series represent, from left to right, ES, EpiSC, STAP,and CD45)). In cluster analysis of the genome-wide transcriptome, STAPcells were closest to ES cells and have substantial similarity toblastocysts in RNA expression, while they are most distant from parentalCD45⁺ cells (data not shown). The situation of X-chromosomalinactivation in STAP cells was intriguing; ˜40% of female STAP cells(d7) showed an inactivated chromosome, while X-chromosomal inactivationwas cancelled in the rest (˜60%)(FIG. 15B).

These findings raised the possibility that the differentiation state ofSTAP cells may represent a new metastable pluripotent state closelyrelated to but distinct from that of ES cells.

Chimera Formation and Germline Transmission in Mice.

Finally, chimera-forming capability of STAP cells was assessed by theblastocyst injection assay. Unlike ES cells, when STAP cells(B6-background) were dissociated into single cells and injected into ICRblastocysts, no chimeric mice carrying the dark coat color were born(Table 4). Since single STAP cells can hardly be maintained in vitro, itwas inferred that cellular dissociation somehow altered their capacity.Therefore, STAP cell clusters were manually cut into small pieces usinga micro-knife under the microscopy, and injected en bloc intoblastocysts (data not shown). With this maneuver, chimeric mice wereborn at a substantial rate and all developed normally (data not shown).Next examined was the tissue contribution of injected STAP cells thatwere generated from CD45⁺ cells of mice constitutively expressing GFP(F1 of C57BL/6GFP crossed with DBA/2 or 129/Sv). A high to moderatecontribution of GFP-expressing cells was seen in chimeric embryosinjected with STAP cell clusters (data not shown).

The contribution rate of GFP⁺ cells in each tissue was analyzed in thesechimeric embryos by FACS. CD45⁺ cell-derived STAP cells contributed toall tissues examined (data not shown). Furthermore, offspring derivedfrom STAP cells were born to the chimeric mice (Table 5). This potentialof STAP cells is important and demonstrates the genuine nature of thesepluripotent cells, since germline transmission is regarded as a strictcriteria for pluripotency as well as genetic and epigenetic normality²².A tetraploid (4N) complementation assay was then performed by injectingcells into 4N blastocysts, which is considered to be the most rigoroustest for developmental potency of the injected cells because theresulting embryos are derived only from these donor cells²³ (data notshown). When injected into 4N blastocysts, CD45⁺ cell-derived STAP cells(from DBA×B6GFP or 129/Sv×B6GFP F1 mice) generated ‘all GFP⁺ embryos’ onE10.5 (data not shown), demonstrating that STAP cells alone weresufficient to construct an entire embryonic structure.

Taken together, these findings explicitly show that STAP cells have thedevelopmental capacity to differentiate into all somatic-cell andgerm-line lineages in the context of embryonic environments.

Discussion

The data described herein have revealed a surprisingly flexibleplasticity that somatic cells latently possess. This dynamic plasticity,even converting into pluripotent cells, emerges when cells aretransiently exposed to strong stimuli that they would not normallyexperience in their living environments.

The conversion from CD45⁺ cells to STAP cells was not substantiallyaffected at least by treatment with HDAC inhibitors (e.g., TricostatinA) or 5-aza-cytidine.

It is demonstrated herein that low-pH treatment substantially reducedthe number of cells in culture. However, in fact, the decrease ofsurviving cells during the first 24 hours was marginal, suggesting thatthis treatment was unlikely to give acute lethal effects on a majorityof cells. Instead, a delayed cell loss gradually occurred during d2-d5.Consistent with this, the data demonstrated that a number of genesinvolved in cellular response to stress and DNA repair²¹ were stronglyinduced in low-pH-experienced oct3/4::GFP⁺ cells on d3, but not incontrol cells cultured in the same medium, suggesting that cellsresponded to the stimulus as life-threatening, or sublethal, stress.Interestingly, their gene expression levels became even higher on d7;therefore, it is intriguing in the future to investigate not only theroles of stress-induced genes for cellular survival, perhaps, but alsotheir possible involvement in the reprogramming process.

Another open question is whether cellular reprogramming may be initiatedspecifically by the low-pH treatment or also by some other types ofsublethal stress such as physical damage, plasma membrane perforation,osmotic pressure shock, growth-factor deprivation, hypoxia and high Ca²⁺medium exposure. Notably, at least some of them, in particular, physicaldamage by rigorous trituration and membrane perforation by streptolysinO, induced the generation of oct3/4::GFP⁺ cells from CD45⁺ cells (FIG.18A). These findings raise the possibility that certain commonregulatory modules, lying downstream of these distantly relatedsublethal stresses, act as a key for releasing somatic cells from thetightly locked epigenetic state of differentiation, leading to theglobal change in the epigenetic regulation. Given that some oct3/4::GFP⁺cells appeared by d2, such a reprogramming mechanism may start tofunction within the first two days.

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Materials and Methods

Tissue Harvesting and Cell Culture.

To isolate mature lymphocytes, spleens derived from 1 week-old GOF miceor ICR mice, were minced by scissors and mechanically-dissociated withpasture pipettes. Dissociated spleens were strain through a cellstrainer (BD Biosciences, San Jose). Collected cells were re-suspendedin DMEM medium and added the same volume of lympholyte (CEDARLANE®,Ontario, Canada), then centrifuged at 1000 g for 15 min Lymphocyteslayer was taken out and attained with CD45 antibody (ab25603, abcam,Cambridge, Mass.). CD45 positive cells were sorted by FACS Aria (BDBiosciences). Then, CD45 positive cells were treated with stresstreatment (pH5.5 solution for 15 min) and plated into B27 mediumsupplemented with 1000 U LIF (Sigma).

Exposure to External Stimuli—Stress Treatment.

To give a mechanical stress to mature cells, pasture pipette were heatedand then stretched to create lumens approximately 50 microns indiameters, and then broken. Mature somatic cells were then trituratedthrough these pipettes for 20 min, and cultured for 7 days. To provide ahypoxic stimulus to mature cells, cells were cultured in a 5% oxygenincubator for 3 weeks. An under nutrition stimulus was provided tomature cells, by culturing the cells in a basal culture medium for 3weeks. High Ca culture concentration was provided to mature cells, byculturing cells in medium containing 2 mM CaCl2 for 7 days. To exposethe mature cells to a physiological stress, they were treated with lowpH (pH5.5) solution, and cultured for 7 days. Also, cells were givenmore serious damage. To create pores in mature cell membranes, cellswere treated with 230 ng/ml SLO (Streptolysin O) (S5265, Sigma) for 2 h,then cultured for 7 days.

Bisulfite Sequence.

For cells procured from GOF mice were dissociated into single cells. GFPpositive cells collected using by FACS Aria™. Genome DNA was extractedfrom SACs and studied. Bisulfite treatment of DNA was done using theCpGenome™ DNA Modification Kit (Chemicon, Temecula, Calif.,http://www.chemicon.com) following the manufacturer's instructions.

The resulting modified DNA was amplified by nested polymerase chainreaction PCR using two forward (F) primers and one reverse (R) primer:Oct4 (F1,GTTGTTTTGTTTTGGTTTTGGATAT;F2,ATGGGTTGAAATATTGGGTTTATTTA;R,CCACCCTCTAACCTTAACCTCTAAC). And Nanog(F1,GAGGATGTTTTTTAAGTTTTTTTT;F2,AATGTTTATGGTGGATTTTGTAGGT;R,CCCACACTCATATCAATATAATAAC). PCR was done using TaKaRa Ex Taq Hot Start Version(RR030A). DNA sequencing was performed using M13 primer with theassistance of GRAS (The Genome Resource and Analysis Unit).

Immunohistochemistry.

Cultured cells were fixed with 4% paraformaldehyde and permeabilizedwith 0.1% Triton X-100/PBS prior blocking with 1% BSA solution (LifeTechnology, Tokyo, Japan). Secondary antibodies were goat anti-mouse or-rabbit coupled to Alexa-488 or -594 (Invitrogen). Cell nuclei werevisualized with DAPI (Sigma). Slides were mounted with SlowFade Goldantifade reagent (Invitrogen).

Fluorescence-Activated Cell Sorting and Flow Cytometry.

Cells were prepared according to standard protocols and suspended in0.1% BSA/PBS on ice prior to FACS. PI™ (BD Biosciences) was used toexclude dead cells. In negative controls, the primary antibody wasreplaced with IgG negative controls of the same isotype to ensurespecificity. Cells were sorted on a BD FACSAria SORP™ and analyzed on aBD LSRII™ with BD FACSDiva™ Software (BD Biosciences).

RNA Preparation and RT-PCR Analysis.

RNA was isolated with the RNeasy™ Micro kit (QIAGEN). Reversetranscription was performed with the SupeSACript III First StrandSynthesis kit (Invitrogen). SYBR Green™ Mix I (Roche Diagnostics) wasused for amplification, and samples were run on a Lightcycler-II™Instrument (Roche Diagnostics).

Animal Studies.

For tumorigenicity studies, cells suspended in 100 ml PBS were injectedsubcutaneously in the flanks of age-matched immunodeficient SCID mice.Mice were sacrificed and necropsied after 6 weeks.

ATP and ROS Assay.

Intercellular ATP level was measured by the ATP Bioluminescence AssayKit HS II™ (Roche) according to supplier's protocol. The luminescenceintensity was measured by using a Gelomax™ 96 Microplate Luminometer(Promega, Madison, Wis.) and the luminescence readings were normalizedby cell count. For measurement of ROS levels, cells were incubated in amedium contain 2 μM dihydroethidium (Molecular Probes) at 37° C. in darkfor 15 minutes. Cells were then washed with PBS and suspended in PBScontaining 0.5% BSA. The fluorescence intensity of 30000 cells wasrecorded with the help of a BD Biosciences LSR II (BD Bioscience, Spark,Md.).

Chimera Mice Generation and Analyses

Production of Diploid and Tetraploid Chimeras.

Diploid embryos were obtained from ICR strain females mated with ICRmales and tetraploid embryos were obtained from BDF1 strain femalesmated with BDF1 males. Tetraploid embryos were produced by theelectrofusion of 2-cell embryos. In this study, because trypsintreatment caused low chimerism, SACs spherical colonies were cut intosmall pieces using a micro-knife under the microscopy, then smallclusters of SACs were injected into day 4.5 blastocyst by large pipette.Next day, the chimeric blastocysts were transferred into day 2.5pseudopregnant females.

In Vitro Differentiation Assay.

Mesoderm Lineage Differentiation Assay.

The stress altered cell masses were collected at 7 days and dissociatedinto single cells, then collected only Oct4-GFP positive cells by cellsorter. Collected cells were DMEM supplemented 20% FCS. Medium wasexchanged every 3 days. After 7-14 days, muscle cells were stained withanti-α-smooth muscle actin antibody (N1584, DAKO). In negative controls,the primary antibody was replaced with IgG negative controls of the sameisotype to ensure specificity.

Neural Lineage Differentiation Assay.

The stress altered cell masses were collected at 7 days and dissociatedinto single cells, then collected only Oct4-GFP positive cells by cellsorter. Collected cells were plated on ornithin-coated chamber slides(Nalge Nunc International) in F12/DMEM (1:1, v/v) supplemented 2% B27(Invitrogen), 10% FCS, 10 ng/ml bFGF (R&D Systems) and 20 ng/m EGF (R&DSystems). Medium was exchanged every 3 days. After 10-14 days, cellswere fixed with 4% paraformaldehyde for 30 minutes at 4° C., washed withPBS containing 0.2% Triton X-100 for 15 minutes at room temperature,incubated with PBS containing 2% FCS for 20 minutes to blocknon-specific reactions, and incubated with anti-βIII Tubuin mousemonoclonal antibody (G7121, Promega) and anti-GFAP mouse monoclonalantibody (AB5804, CHEMICON). In negative controls, the primary antibodywas replaced with IgG negative controls of the same isotype to ensurespecificity.

Hepatic Differentiation Assay.

The stress altered cell masses were collected at 7 days and dissociatedinto single cells, then collected only Oct4-GFP positive cells by cellsorter. Collected cells were plated on chamber 2 well slide glass (NalgeNunc International) in Hepatocyte culture medium composed of 500 mLhepatocyte basal medium (Lonza, Wuppertal, Germany), 0.5 mL ascorbicacid, 10 mLBSA-FAF (fatty acid free), 0.5 mL hydrocortisone, 0.5 mLtransferrin, 0.5 mL insulin, 0.5 mL EGF, and 0.5 mLgentamycin-amphotericin (GA-1000; all from Lonza) supplemented with 10%FCS, 1% Penicillin/Streptomycin (Sigma). Differentiated cells weredetected by immunohistochemistory using following antibodies;anti-α-fetoprotein mouse monoclonal antibody (MAB1368, R&D System) andanti-Cytokeratin 7 mouse monoclonal antibody (ab668, abcam). In negativecontrols, the primary antibody was replaced with IgG negative controlsof the same isotype to ensure specificity.

In Vivo Differentiation Assay:

The stress altered cell masses were collected at 7 days and dissociatedinto single cells, then collected only Oct4-GFP positive cells by cellsorter. Collected cells were re-suspended in 50 ul of DMEM with 10% FBS.This solution was seeded onto a sheet 3×3×1 mm, composed of a non wovenmesh of polyglycolic acid fibers, 200 microns in diameter, and implantedsubcutaneously into the dorsal flanks of a 4 week old NOD/SCID mouse.Four weeks later the implants were harvested, and analyzed usingimmunohistochemical techniques. The implants were fixed with 10%formaldehyde, embedded in paraffin, and routinely processed into 4-μmthick. Sections were stained with hematoxylin and eosin. Endodermtissues were identified with endoderm marker anti-α-fetoprotein mousemonoclonal antibody (MAB1368, R&D System). Ectoderm tissues wereidentified with anti-βIII Tubulin mouse monoclonal antibody (G7121,Promega). Mesoderm tissues were identified with anti-α-smooth muscleactin antibody (N1584, DAKO). In negative controls, the primary antibodywas replaced with IgG negative controls of the same isotype to ensurespecificity.

TCRβ Chain Rearrangement Analysis.

gDNA was extracted from SACs and tail tips from chimeric mice generatedwith SACs derived from CD45 positive cells. PCR was performed with 50 nggDNA using following primers. (5 ‘-GCACCTGTGGGGAAGAAACT-3’ and 5‘-TGAGAGCTGTCTCCTACTATCGATT-3’) Amplified DNA was electrophoresed with1.5% agarose gel.

Genotyping of Chimera Mice.

gDNA was extracted from tail tips from 4N chimeric mice.

Genotyping was performed using following primers. (GFP:F-AGAACTGGGACCACTCCAGTG and R-TTCACCCTCTCCACTGACAGATCT. IL-2:F-CTAGGCCACAGAATTGAAAGATCT and R-GTAGGTGGAAATTCTAGCATCATCC)

The optimum culture conditions for maintaining stress altered Oct4expressing cells were then determined Several previously describedculture media, including: ES establishment culture medium, 3i¹⁶ andACTH¹⁷, ES culture condition, ES-LIF¹⁸, Oct4-expressing primitive neuralstem cell culture condition, B27-LIF¹⁹, and EpiSCs culture condition²⁰,were examined. Cells were plated into each medium, and GFP expressedcolonies were counted (FIG. S1C). The medium B27-LIF appeared to be themost effective in generating GFP expressing spherical colonies.Therefore we utilized B27-LIF medium for culture of the treated cells.

In order to examine whether SACs generated from cells procured fromvarious tissues had different differentiation tendency, SACs weregenerated from various tissues derived from F1 GFP mice, injected theminto ICR blastocysts. Then, using FACS, the contribution ratio of eachtissue in the generated chimeric mice was analyzed. It was found thatSACs derived from any tissue contributed to chimeric mouse generation(data not shown). In addition, the contribution ratio to skin, brain,muscle, fat, liver and lung was analyzed in chimeric mice generatedusing SACs derived from various tissues. SACs, derived from any tissue,contributed to generate tissues representative of all three germ layers,and no differentiation tendency was observed (data not shown).

TABLE 4 Generation of chimeric mice from SACs No. of No. of chimericCell Culture fertilized mice obtained Mouse preparation period ofembryos No. High strain for injection SACs injected offspring Totalcontribution** BDF1 Cluster 7 day 58  48* 16 4 129B6F1 Cluster 7 day 9864 20 6 GOF Cluster 7 day 73 35 24 2 GOF Cluster 10 day  35 20  4 0 *Allfetuses were collected at 13.5 dpc to 15.5 dpc and the contribution rateof SACs into each organs was examined by FACS **The contribution of SACsinto each chimera was scored as high (>50% of the coat color of GFPexpression)

TABLE 5 Production of offspring from SACs via germ lines transmission ofchimeric mice SAC contribution in Mouse strain No. No. of offspringchimeras body of host total with GFP or Pair ID. Male Female blastocystpups black eyes (%) No. 1 High Medium ICR 9 5 (56) No. 2 High Nonchimera ICR 11 4 (36) 14 4 (29) No. 3 Medium Low ICR 9 0 10 0 13 0 No. 4Medium Medium ICR 4 2 (50) 10 6 (60) 11 7 (64) No. 5 Medium MediumBALB/c 9 4 (44) 5 3 (60)

Example 3

Without wishing to be bound by theory, the methods described herein arecontemplated to be activating a process related to apoptosis, orcontrolled cell death. Mild injury to cells can induce the activation ofrepair genes. Severe injury to cells can activate a previously undefinedsurvival mechanism. It is contemplated that when cells are exposed to asignificant stress, such as the stresses described herein, the cellularcomponents (e.g. mitochondria, vesicles, nuclei, ribosomes, endoplasmicreticulum, exosomes, endosomes, cell membranes, mitochondria, lysosomes,ATP, proteins, enzymes, carbohydrates, lipids, etc) are released fromthe damaged cells into a “cellieu.” Data described herein indicate thatthis “cellieu” can be capable of reconstituting and/or promoting thesurvival of cells. It is additionally contemplated, without wishing tobe bound by theory, that mitochondria (and other organelles) are able todirect the reconstitution of the cells. Because of the small size,simplicity, ability to direct cell differentiation, and prokaryotic-likenature, mitochondria may survive stresses that prove lethal to theparent cell. Mitochondria can be released from the cell free,encapsulated in a membrane, and/or bound to other cellular components.

Alternatively, without wishing to be bound by theory, the nuclei canremain intact, encapsulated in a cell membrane which can comprise somemitochondria. These damaged cells with very little cytoplasm and veryfew organelles, which have lost the epigenetic control of the nucleus,can then interact and possibly fuse with organelles that have beenextruded. This provides cells with the subcellular components necessaryfor growth and replication but the cells have lost epigenetic control,and therefore a more primitive (e.g. more pluripotent) state is induced.

Example 4 Developmental Potential for Embryonic and Placental Lineagesin Reprogrammed Cells with Acquired Pluripotency

In general, the fates of postnatal somatic cells are fixed and do notchanged unless they undergo nuclear transfer^(1,2) or geneticmanipulation with key transcription factors³. As demonstrated herein,the inventors have discovered the unexpected phenomenon of somatic cellreprogramming into pluripotent cells by sublethal stimuli, calledstimulation-trigged acquisition of pluripotency (STAP)⁴. Also describedherein is the demonstration that reprogrammed STAP cells exhibit aunique differentiation capacity that is distinct from ES cells. STAPcells can contribute not only to embryonic tissues but also to theplacental system, as seen in a blastocyst injection assay. Theirefficacy for placental contribution was further strengthened by culturewith FGF4. Conversely, when cultured for additional passages in ES cellmaintenance medium, STAP cells, which originally showed a limitedself-renewal ability, generate robustly proliferating cell lines thatexhibit ES cell-like, but not trophoblast-like, characteristics. Thesealtered STAP cells (STAP stem cells) gave birth to mice in a tetraploidcomplementation assay⁵, but no longer contribute to placental tissues.Thus, STAP cells, unlike iPS cells, may represent a novel metastablestate of pluripotency⁶ that differs from that of ES cells. STAP stemcell technology may offer a versatile, powerful resource fornew-generation regenerative medicine.

Described herein is an intriguing phenomenon of cellular fateconversion: somatic cells regain pluripotency after experiencingsublethal stimuli such as a low-pH exposure⁴. When splenic CD45⁺ cells(including committed T cells) are exposed to pH5.7 for 30 min andsubsequently cultured in the presence of LIF, a substantial portion ofsurviving cells start expressing the pluripotent ell marker Oct3/4 atday 2 (d2). By d7, pluripotent cell clusters form with a bona fidepluripotency marker profile and competence for three germ-layerdifferentiation (e.g., as shown by teratoma formation). These STAP cellscan also efficiently contribute to chimeric mice and undergo germ-linetransmission in a blastocyst injection assay. While thesecharacteristics resemble those of ES cells, STAP cells appear to differfrom ES cells, at least, in their limited capacity for self-renewal(typically, 3-5 passages at maximum) and in their vulnerability todissociation culture⁴.

In the present example, the inventors further investigated the uniquenature of STAP cells, focusing on their differentiation potential intotwo major categories of cells in the blastocyst⁷⁻⁹: inner cell mass-type(or ES cell-like) cells and trophoblast/placental-lineage cells after ablastocyst injection assay revealed an unexpected finding. In general,progeny of injected ES cells are found in the embryonic portion of thechimera, but rarely in the placental portion⁷ (data not shown).Surprisingly, injected STAP cells contributed not only to the embryo butalso to the placenta and extraembryonic membranes (FIG. 22). Thisdual-lineage contribution was observed in roughly 60% of the chimericembryos.

This finding prompted the investigation of the trophoblasticdifferentiation capacity of STAP cells. It is known that trophoblasticcell lines (trophoblast stem cells; TS cells)^(8,9) can be derived inprolonged adhesion culture of blastocysts in the presence of FGF4. WhenSTAP cell clusters were cultured under the same conditions (FIG. 23A;one cluster per well in a 96-well plate), spheroid STAP cell clustersgradually disappeared, and cells with a flat appearance distinct fromSTAP cells grew out and formed colonies by d7-d10 (data not shown).Unlike STAP cells, which have a high level of oct3/4::GFP expression,these flat cells (adhering to the plate bottom) exhibited moderate GFPsignals at day 7 of culture with FGF4 (data not shown). Immunostainingshowed that FGF4-induced (F4I) cells strongly expressed thetrophoblastic markers¹⁰⁻¹² Integrin alpha 7 and Eomesodermin (data notshown) in addition to moderate levels of oct3/4::GFP. The expression ofNanog was detectable but quite low (data not shown). Consistent withthis, qPCR analysis indicated that F4I cells expressed substantiallevels of trophoblast-lineage marker genes (e.g., cdx2), while theirexpression of oct3/4 and nanog was lower than that seen in parental STAPcells (FIG. 23B). These F4I cells could be expanded efficiently bypassaging with trypsin digestion every third day and they remainedstable for more than 30 passages in the presence of FGF4 (in itsabsence, they stopped proliferation). While this establishment andexpansion could be done both on MEF cells and on the gelatin-coatedbottom, those cultured on the MEF feeder tended to show clearerepithelial appearance (data not shown).

In the blastocyst injection assay, the placental contribution of F4Icells was frequently observed (50-60%) (data not shown). In the chimericplacentae, F4I cells typically contributed to ˜10% of total placentalcells (FIG. 23C, lanes 1-3; note that control ES cells gave nosubstantial placental contribution, lanes 4-6). These findings suggestthat STAP cells have the competence to generate TS-like cells throughFGF4 treatment, at least in the light of trophoblast marker expressionand placental contribution. Since this type of derivation into TS-likecells is not common with ES cells (unless genetically manipulated)¹¹,such competence may represent another feature of STAP cells that isdistinct from ES cells.

On the other hand, F4I cells derived from STAP cells may also possessdifferent characteristics from blastocyst-derived TS cells. First,unlike conventional TS cells¹³, F4I cells expressed a moderate level ofoct3/4 (data not shown). Furthermore, unlike TS cells,blastocyst-injected F4I cells also contributed to the embryonic portions(in all cases that involved chimeric placentae), although the extent ofcontribution was generally low (data not shown).

Collectively, these observations indicate that the STAP cell populationis qualitatively different from ES cells with respect to theircompetence for placental differentiation.

With this in mind, the differentiation into the embryonic lineage,another cell type present in the blastocyst was investigated. Unlike EScells, STAP cells have a limited self-renewal capacity and cannot beexpanded from single cells. STAP cells could not be maintained for morethan 5 passages (even with partial dissociation culture of clusters) inconventional LIF-containing media (including the B27+LIF medium used inthe STAP cell establishment). However, an ACTH-containing medium withLIF¹⁵ (ACTH medium, hereafter) had relatively good supporting effects onthe growth speed of STAP cell colonies (data not shown). When culturedin this medium on a MEF feeder or gelatin in ACTH medium (FIG. 24A),some portion of STAP cell clusters (typically found in 20-50% of wellsin single cluster culture using 96-well plates) continued to grow (datanot shown). These growing colonies were similar to those of mouse EScells and expressed a high level of oct3/4::GFP. Unlike parental STAPcells, the cells in these expanded colonies, after culturing in thismedium for seven days, became resistant to dissociation and could bepassaged as single cells (data not shown). In contrast to STAP cells,these altered cells could be expanded exponentially, up to at least 120days of culture (FIG. 24B), like ES cells. This enhanced expandabilitywas not accompanied by chromosomal abnormality, as shown by multi-colorFISH analysis¹⁶ (data not shown). After the seven-day expansion, thecells grew and could be maintained in any of the ES cell media tested,while this initial 7-day expansion was most efficiently done with ACTHmedium (for instance, colonies formed slowly and less frequently in 3imedium¹⁷; data not shown).

Hereafter, the proliferative cells derived from STAP cells are referredto as STAP stem cells. Unlike STAP cells, STAP stem cells did notproduce TS-like cells in culture with FGF4 (data not shown). Throughimmunostaining, it was found that X-chromosomal inactivation¹⁸, whichwas found in a substantial proportion of female STAP cells (ref), wasnot observed in STAP stem cells any longer (data not shown). STAP stemcells expressed various RNA (FIG. 24C) and protein (data not shown)markers for ES cells. The DNA methylation levels at the oct3/4 and nanogloci, which become demethylated upon the conversion from CD45⁺ to STAPcells, remained low (FIG. 24D). In differentiation culture¹⁹⁻²¹, STAPstem cells generated ectodermal, mesodermal and endodermal derivatives(data not shown). These findings demonstrate that STAP stem cellsexhibit features indistinguishable from those of ES cells.

Consistent with this, STAP stem cells, even after multiple passages,could form teratomas (data not shown) and, by blastocyst injection,efficiently contribute to chimeric mice (data not shown). The remarkableefficacy of STAP stem cells in their embryonic contribution wasexplicitly demonstrated by the fact that in the tetraploid complementaryassay⁵, these cells could give birth to mice capable of growing toadults and even generating offspring (data not shown). Given that eightindependent lines of STAP stem cells reproducibly showed this ability(note that such complete complementation is often difficult even withcommonly used ES cell lines), we infer that STAP cells, which originatefrom adult somatic cells, could be an attractive source for derivationof pluripotent stem cell lines, equivalent to (or maybe superior to)blastocysts themselves in this aspect.

Importantly, unlike STAP and F4I cells, STAP stem cells appear to havelost their ability to contribute to placental tissues (data not shown),whereas they gave rise to various tissues in the chimeras (FIGS.25A-25B). Therefore, the difference between STAP cells and STAP stemcells is not merely limited to self-renewal activities, but alsoinvolves the loss of competence to differentiate into placentallineages.

These findings indicate a unique pluripotent state of STAP cells. Whilethe inability to clone STAP cells from single cells (described above)hinders commitment analysis at the single-cell level, it is worth notingthat the STAP procedure can convert somatic cells into a pluripotentcell population with competence for both embryonic and placentallineages. hi-depth understanding of the differentiation state of STAPcells is an important topic for future study. In particular, it will beinteresting to investigate whether STAP cells represent a more immaturestate than ES cells, as suggested by their competence for placentallineages, which resembles embryonic cells at the morula stage. A recentstudy has reported that conventional ES cell culture also contains avery minor population of Oct3/4⁻ cells with a distinct characterresembling the feature of very early-stage embryos²². STAP cells mayhave a similar metastable state allowing the dual-competence capacitybut, unlike ES cells, this is found in a majority of the cellpopulation.

It is demonstrated herein that STAP cells have a capacity fortransformation into ES-like pluripotent stem cell lines. It is worthnoting that STAP cells (from female mice) are somewhat mosaic inX-chromosome inactivation; the inactivation disappears in ˜40% of STAPcells⁴, while the rest maintain it. In ES cells, by contrast, bothX-chromosomes are reproducibly activated. Interestingly, afterderivation, STAP ‘stem’ cells show no X-chromosome inactivation, like EScells, suggesting that epigenetic control in parental STAP cells issimilar but not identical to that of mouse ES cells, also in this sense.

The present results demonstrate an unexpected ‘spontaneous convertingability’ of committed somatic cells to reprogram their own fates intonaïve cells upon exposure to sublethal stimuli. This raises numerousintriguing and profound biological questions including those describedabove. On top of those, this newly discovered STAP phenomenon canrevolutionize methodologies in stem cell medicine. It is contemplatedthat the generation of various types of tissues can be permitted bysteered differentiation from STAP cells, or STAP stem cells, that arederived from somatic cells without gene transfer (which may increase therisk of cancerous transformation). Moreover, unlike iPS cell conversion,STAP conversion occurs at a significantly high frequency and proceeds bycertain endogenous programs that are triggered by strong stimuli such asa low-pH exposure. Since STAP stem cells, like ES cells, are easilyexpandable and clonable, they would be more suitable than STAP cells forlarge-scale generation of medically useful tissues under strict qualitycontrol. In our preliminary study, the inventors have succeeded indemonstrating efficient differentiation of STAP stem cells into retinalprogenitors²³, cortical progenitors²⁴ and beating cardiomyocytes²⁵ (datanot shown).

Methods

Cell Culture.

STAP cells were generated from CD45⁺ cells by a transient exposure tolow-pH solution, followed by culture in B27+LIF medium (Obokata et al,2013; co-submitted). For F4I cell line establishment, STAP cell clusterswere transferred to FGF4-containing TS medium on MEF feeder cells in96-well plates. The cells were subjected to the first passage duringd7-d10 using a conventional trypsin method. For the establishment ofSTAP stem (STAPS) cell lines, STAP spheres were transferred toACTH-containing medium on a MEF feeder or gelatin-coated dish. Four toseven days later, the cells were subjected to the first passage using aconventional trypsin method, and suspended cells were plated in ESmaintain medium containing 5% FCS and 1% KSR.

Chimera Mice Generation and Analyses.

For injection of STAP stem cells, F4I cells and ES cells, a conventionalblastocyst injection method was used. For STAP cell injection, STAP cellclusters were injected en bloc, because trypsin treatment caused lowchimerism. STAP spherical colonies were cut into small pieces using amicro-knife under the microscopy, then small clusters of STAP colonywere injected into day-4.5 blastocyst by large pipette. Next day, thechimeric blastocysts were transferred into day-2.5 pseudopregnantfemales. Tetraploid embryos were produced by electrofusion of 2-cellembryos.

In Vitro and In Vivo Differentiation Assay:

Teratoma formation was examined by injecting 1×10⁵ cells of STAPS cellssubcutaneously into the dorsal flanks of 4 week-old NOD/SCID mice. Invitro neural differentiation was induced by the SDIA and SFEBqmethods^(24,26). In vitro endomesodermal differentiation²⁵ was inducedby culturing STAPS cell aggregate with growth factors (Activin) or 10%FCS.

Karyotype Analysis.

Subconfluent STAPS cells were arrested in metaphase by colcemid andsubjected to multicolor FISH analysis (M-FISH). Mousechromosome-specific painting probes were combinatorially labeled usingseven different fluorochromes and hybridized as previously described(Jentsch et al., 2003).

Cell Culture.

STAP cells were generated from CD45⁺ cells, followed by culture inB27+LIF medium for 7 days, as described (Obokata et al, 2013;co-submitted). For F4I cell line establishment, STAP cell clusters weretransferred to FGF4-containing TS medium on MEF feeder cells in 96-wellplates. The cells were subjected to the first passage during d7-d10using a conventional trypsin method. Subsequent passages were performedevery third day.

For STAP stem (STAPS) cell line establishment, STAP spheres weretransferred to ACTH-containing medium on MEF feeder cells. Four to sevendays later, the cells were subjected to the first passage using aconventional trypsin method, and suspended cells were plated in ESmaintain medium containing 5% FCS and 1% KSR. Subsequent passaging wasperformed every second day.

Chimera Mice Generation and Analyses.

For the production of diploid and tetraploid chimeras, diploid embryoswere obtained from ICR strain females mated with ICR males andtetraploid embryos were obtained from BDF1 strain females mated withBDF1 males. Tetraploid embryos were produced by electrofusion of 2-cellembryos. For injection of STAP stem cells, F4I cells and ES cells, aconventional blastocyst injection method was used. For injection of STAPstem cells, F4I cells and ES cells, a conventional blastocyst injectionmethod was used. For STAP cell injection, STAP cell clusters wereinjected en bloc, because trypsin treatment caused low chimerism. STAPspherical colonies were cut into small pieces using a micro-knife underthe microscopy, then small clusters of STAP colony were injected intoday-4.5 blastocyst by large pipette. Next day, the chimeric blastocystswere transferred into day-2.5 pseudopregnant females.

In Vitro and In Vivo Differentiation Assay:

1×10⁵ cells of STAP-S cells were injected subcutaneously into the dorsalflanks of 4 week-old NOD/SCID mice. Six weeks later, the implants wereharvested, and histologically analyzed. The implants were fixed with 10%formaldehyde, embedded in paraffin, and routinely processed into 4-μmthick. Sections were stained with hematoxylin and eosin.

In vitro neural differentiation was induced by the SDIA and SFEBqmethods. In vitro endomesodermal differentiation was induced byculturing STAPS cell aggregate with growth factors (Activin) or 10% FCS.

Immunostaining.

Cells were fixed with 4% PFA for 15 min and, after permeabilization with0.5% Triton X-100 and then incubated with primary antibodies: antiH3K27me3 (Millipore; 1:300), anti-Oct3/4 (Santa Cruz Biotechnology;1:300), anti-Nanog (eBioscience; 1:300), anti-KLF2/4 (R&D System;1:300), and anti-Esrrβ (R&D System; 1:300). After overnight incubation,bounded antibodies were visualized with a secondary antibody conjugatedto Alexa546 (Molecular Probes). Nuclei were stained with DAPI (MolecularProbes).

RNA Preparation and RT-PCR Analysis.

RNA was isolated with the RNeasy™ Mini kit (QIAGEN). Reversetranscription was performed with the SupeSACript III First StrandSynthesis kit (Invitrogen). Power SYBR™ Green Mix (Roche Diagnostics)was used for PCR amplification, and samples were run on aLightcycler-II™ Instrument (Roche Diagnostics).

Karyotype Analysis.

Karyotype analysis was performed by Multicolor FISH analysis (M-FISH).Subconfluent STAPS cells were arrested in metaphase by colcemid (finalconcentration 0.270 μg/ml) to the culture medium for 2.5 h at 37° C. in5% CO2. Cells were washed with PBS, treated withtrypsin/ethylenediaminetetraacetic acid (EDTA), resuspended into cellmedium and centrifuged for 5 min at 1200 rpm. To the cell pellet in 3 mlof PBS, 7 ml of a prewarmed hypotonic 0.0375 M KCl solution was added.Cells were incubated for 20 min at 37° C. Cells were centrifuged for 5min at 1200 rpm and the pellet was resuspended in 3-5 ml of 0.0375 M KClsolution. The cells were fixed with methanol/acetic acid (3:1; vol/vol)by gently pipetting. Fixation was performed four times prior tospreading the cells on glass slides. For the FISH procedure, mousechromosome-specific painting probes were combinatorially labeled usingseven different fluorochromes and hybridized as previously described(Jentsch et al., 2003). For each cell line, 9-15 metaphase spreads wereacquired by using a Leica DM RXA RF8 epifluorescence microscope (LeicaMikrosysteme GmbH, Bensheim, Germany) equipped with a Sensys CCD camera(Photometrics, Tucson, Ariz.). Camera and microscope were controlled bythe Leica Q-FISH software (Leica Microsystems hanging solutions,Cambridge, United Kingdom). Metaphase spreads were processed on thebasis of the Leica MCK software and presented as multicolor karyograms.

Bisulfate Sequence.

Genome DNA was extracted from STAPS cells. Bisulfate treatment of DNAwas performed using the CpGenome DNA Modification Kit (Chemicon,Temecula, Calif., http://www.chemicon.com) following the manufacturer'sinstructions.

The resulting modified DNA was amplified by nested polymerase chainreaction PCR using two forward (F) primers and one reverse (R) primer:oct3/4 (F1, GTTGTTTTGTTTTGGTTTTGGATAT (SEQ ID NO:73);F2,ATGGGTTGAAATATTGGGTTTATTTA (SEQ ID NO:74);R,CCACCCTCTAACCTTAACCTCTAAC (SEQ ID NO: 75)). And nanog(F1,GAGGATGTTTTTTAAGTTTTTTTT (SEQ ID NO: 76);F2,AATGTTTATGGTGGATTTTGTAGGT (SEQ ID NO: 77);R,CCCACACTCATATCAATATAATAAC (SEQ ID NO: 78)). PCR was done using TaKaRaEx Taq Hot Start Version (RR030A). DNA sequencing was performed usingM13 primer at the Genome Resource and Analysis Unit, RIKEN CDB.

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TABLE 6 Establishment of pluripotent cell lines from STAP No. of No.established Pluripotency test Mouse strain used wells*. cell lines (%)(chimera formation) C57BL/6(GOF) 29 29 (100) Yes 129B6F1(GFP) 16 12(75)  Yes 129/Sv (GFP) 2  2 (100) Yes *Each well contained 1-4 piece ofSTAP

TABLE 7 Production of STAPS mouse from FLS cell lines by tetraploidcomplementation method No. of No. of Cell line chimera Callus No. of No.of Germ line name embryos mice survived adulthood transmission FLS-1 317 7 4 Yes FLS-2 29 3 2 2 Yes FLS-3 46 8 8 4 Yes FLS-4 46 9 8 2 Yes FLS-521 10  9 5 Yes FLS-6 12 4 4 4 Yes FLS-7 21 6 3 3 Yes FLS-8 22 5 2 2 YesSubtotal 228 52 (22.8) 43 (82.7) 26 (60.5) Cont-1 8 5 5 4 Yes Cont-2 215 5 4 Yes Cont-3 21 3 1 0 — Subtotal 50 13 (26.0) 11 (84.6)  8 (72.7) *Offspring were mixed and fostered into same mother due to the lack ofenough number of foster mother

TABLE 8 Production of chimera mice from FLS cell lines using diploidembryo Cell line No. of chimera No. of No. of chimera Germ line nameembryos offspring Total Very high High low transmission FLS-1 16 7 6 2 31 Yes FLS-2 17 13 9 2 2 5 Yes FLS-3 32 16 12 6 4 2 Yes FLS-4 20 5 4 1 12 Yes FLS-5 21 5 4 3 0 1 Yes FLS-6 21 13 7 3 3 1 Yes FLS-7 32 14 11 5 51 Yes FLS-8 32 12 8 3 2 3 Yes Subtotal 191 84 62 (73.8) Cont-1 16 9 9 62 1 Yes Cont-2 18 12 8 3 2 3 Yes Cont-3 18 11 4 0 1 3 Yes Subtotal 52 3221 (65.6)

TABLE 9 Cell characteristics ES STAP STAPS Self-renewal unlimitedlimited unlimited Chimera fetus fetus fetus contribution placenta yolksac Dissociation tolerant intolerant tolerant tolerance

Example 5 Protocol for Generating STAP Cells from Mature Somatic Cells

Described herein is an improved protocol for generating STAP cells,regardless of the cells type being studied. The protocol below is animprovement over the methods described in our Jan. 31, 2014 articlepublished in Nature (Obokata et. AL, Stimulus triggered fate conversionof somatic cells into pluripotency. Nature 505. 641-647, 2014) andprovides, e.g. increased efficiency and yield. The protocol is extremelysimple, but will vary slightly, if starting with tissue rather than acell suspension. It also will vary depending upon the cell type ortissue with is used as a starting material.

In some embodiment, do not skip any steps. In some embodiments,triturate the cell suspension for a minimum of 30 minutes, until thesuspension can be easily triturated up and down the reduced borepipettes of the smallest orifices. The protocol is first described whenstarting with a suspension of cells, and then describe additional stepsnecessary when starting with a soft tissue.

Generating STAP Cells when Starting with a Suspension of Mature SomaticCells:

A1. Live somatic cells should be suspended in a centrifuge tube, andthen centrifuged at 1200 rpm for 5 minutes. Note: Trypsin-EDTA, 0.05%(Gibco: 25300-054) can be added to the tissue culture dish containingcells, for 3-5 minutes, to release adherent cells to be added to thecentrifuge tube.A2. Aspirate the supernatant down to the cell pellet.A3. Resuspend the resulting pellet a concentration of 1×10⁶ cells/ml inof Hanks Balanced Saline Solution (HBSS Ca⁺Mg⁺ Free: Gibco 14170-112) in50 ml tube. For example, the pellet can be resuspended in 2-3 mL HBSS ina 50 mL tube.A4. Precoat a standard 9″ glass pipette with media (so the cells do nostick to the pipette—an exemplary pipette is the Fisher brand 9″Disposable Pasteur Pipettes: 13-678-20D). Triturate the cell suspensionin and out of the pipette for 5 minutes to dissociate cell aggregatesand any associated debris. This can be done with a fair amount of force.A5. As a final step in the trituration process, make two fire polishedpipettes with very small orifices as follows:

-   -   Heat the standard 9″ glass pipette over a Bunsen burner and then        pull and stretch the distal (melting) end of the pipette, until        the lumen collapses and the tip breaks off, leaving a closed,        pointed glass tip. Wait until the pipette cools, and then break        off the closed distal tip until a very small lumen is now        identifiable. Repeat this process with the second pipette, but        break the tip off a little more proximally, creating a slightly        larger distal lumen. The larger lumen should be about 100-150        microns in diameter, while the other pipette should have a        smaller lumen of about 50-70 microns.        Now triturate the cell suspension through the pipette with the        larger lumen for 10 minutes. Follow this with trituration        through the pipette having the smaller lumen (50-70 microns) for        an additional 15 minutes. Continue to triturate the suspension        until it passes easily up and down the fire polished pipette of        the smaller bore. Precoat each pipette with media. Also, during        trituration, aspirating air and creating bubbles or foam in the        cell suspension is to be avoided.        A6. Add HSBB to the suspension to a total volume of 20 ml,        centrifuge at 1200 rpm for 5 minutes and then aspirate the        supernatant.        A7. Resuspend the cells in HBSS at a pH of 5.4, at cell        concentration of 2×10⁶ cells/ml, then place in an incubator at        37° C. for 25 minutes. The pH of the HBSS will increase with the        addition of the cell suspension, so an HBSS solution of lower        than the desired final pH of 5.6 can be used.    -   When making the solution acidic, mildly pipette it using a 5 ml        pipette for 10 seconds immediately after adding the acid to the        Hanks Solution. HBSS has a very weak buffering capacity, so any        solution transferred from the supernatant of the previous        suspension will affect the pH of the HBSS drastically. The        instructions below will show how to create HBSS with the optimum        pH of 5.6-5.7 for STAP cell generation according to this        experimental embodiment.        First, titrate the pH of pre-chilled HBSS (at 4 degrees C.) with        12N HCl to a pH of 5.6. This is done by slowly adding 11.6 ul of        12 N HCl to 50 ml of HBSS. After confirming this pH, sterilize        the solution by filtering through a 0.2 micron syringe filter or        bottle top filter of, into a new sterile container for storage.        Please confirm the final pH of 5.6-5.7 through an initial test        experiment with an appropriate number of cells. Because the pH        of the HBSS is so important, the pH of the solution be checked,        re-titrated and re-sterilized prior to each use.        A8. After 25 minutes in the acid bath, centrifuge the suspension        at 1200 rpm for 5 minutes.        A9. Aspirate the supernatant and resuspend the resulting pellet        in 5 ml of what is termed herein “sphere media” (DMEM/F12 with        1% Antibiotic and 2% B27 Gibco 12587-010) and place at a        concentration of 10⁵ cells/cc, within a non-adherent tissue        culture dish in the presence of the following supplements: b-FGF        (20 ng/ml), EGF (20 ng/ml), heparin (0.2%, Stem Cell        Technologies 07980). LIF (1000 U) should be added if the cells        are murine). In some embodiments, supplements such as bFGF, EGF        and heparin may not be necessary.        After the cells are placed in tissue culture dishes, they can be        gently pipetted using a 5 ml pipette, twice/day for 2 minutes,        for the first week, to discourage them from attaching to the        bottom of the dishes. In some embodiment, this can promote good        sphere formation. Add sphere media containing the supplements        described every other day. (Add 1 ml/day to a 10 cm culture        dish, or 0.5 ml/day to a 6 cm dish)

B. Generating STAP Cells when Starting with Soft Tissues that ContainMany RBCs.

B1. Place the excised, washed sterile organ tissue into an 60 mm petridish containing 50 ul of collagenase. (The spleen may not need to beexposed to any digestive enzymes.) It is contemplated herein thatdifferent types of collagenase or enzymes are better for digestion ofdifferent organ tissues.B2. Mince and scrape the tissue for 10 minutes using scalpels andscissors to increase surface area that is exposed to the collagenase,until the tissue appears to become gelatinous in consistency.B3. Add an additional 450 ul of collagenase to the dish and place in anincubator/shaker for 30 minutes at 37° C. at 90 RPM.B4. Add 1.5 ml of HBSS to the dish (yielding a total volume of 2.0 ml)and then aspirate the entire contents via a 5 ml pipette and place intoa 50 ml tube.B5. Now proceed to triturate as previously described above (step A4-5)when starting with mature somatic cells.B6. After trituration is completed (through step A5 when using a culturedish of mature somatic cells), add 3 ml of HBSS, yielding a volume of 5ml, to the 15 ml tube and then slowly add 5 ml of Lympholyte to thebottom of the tube to create a good bilayer. In some embodiments, mixingof the two solutions should be avoided.B7. Centrifuge this tube at 1000 g for 10 min Rotate the tube 180° andrecentrifuge at 1000 g for an additional 10 min. This will cause theerythrocytes to form a pellet at the bottom of the tube.B8. Using a standard 9″ glass pipette aspirate the cell suspensionslayer between HBSS and Lympholyte and place in a new 50 ml tube.B9. Add HSBB to the suspension to a total volume of 20 ml of HBSS andthen thoroughly mix the suspension by pipetting via a 5 ml pipette for 1minutes.B10. Centrifuge the solution at 1,200 rpm for 5 minutes and aspirate thesupernatant.B11. For the next steps see A7-9 as described in this Example.

Example 6 The Restoration by Adult STAP Stem Cells of NormalHyperalgesic Responses Diminished by the Chemical Ablation of NK-1Expressing Neurons in the Rat Spinal Cord

Spinal cord injury presents with a complex of often chronic neurologicalsensory abnormalities, including numbness, paraesthesias and pain.Understanding the mechanisms underlying any one of these, and developingeffective therapeutics is complicated by the broad pathological changesresulting from these traumatic injuries. Described herein is a veryspecific cytological injury in the spinal cord to produce a limited butwell-defined sensory deficit which has then been reversed by implantingstressed adult stem cells (altered by the Stimulus-Triggered Acquisitionof Pluripotency, STAP).

The highly specific cytotoxin SSP-SAP (20 uL, 1 uM) was injected intothe intrathecal (i.t.) space of the male rat spinal cord in order toablate a large majority of the neurokinin-1 receptor (NK1R)-expressingneurons. Two to 3 weeks later the normally robust hyperalgesic responsesto injection of capsaicin (10 uL, 0.1%) in the plantar hindpaw,consisting of mechanical hyperalgesia to stimulation by von Freyfilaments and thermal hyperalgesia appearing as a shortening of thelatency of withdrawal to a radiant heat source, were almost absent.Subsequent i.t. injection of the STAP stem cells, either as a suspensionof individual cells or as spherical aggregates of cells, led to a slowrestoration, over the next 1-2 weeks, of capsaicin-induced mechanicaland thermal hyperalgesia. The restored response had the same amplitudeand time-course as the native, pre-ablation response, and was fullyinhibited by i.t. injection of the NK-1R antagonist L-733,060 (at 300uM). Immunocytochemistry of the lumbar spinal cord from rats withrestored hyperalgesic functions revealed staining of NK-1R throughoutthe dorsal horn. It thus appears that STAP stem cells can restore normalfunction after specific spinal neuronal loss and present a model for atherapeutic approach to spinal cord injury.

Adult male S-D rats were first handled for 4-5 days to familiarize themwith the test arena, to minimize stress-induced analgesia, and to obtainNaïve and initial Baseline behavioral data. Tactile responsiveness wasdetermined by probing the plantar surface of one hindpaw with a 15 g vonFrey filament (VFH), 10 times every 3 secs. Baseline sensitivity, withno treatments and no capsaicin equaled ˜1-2 paw withdrawals per 10probes. Thermal sensitivity was indicated by the latency for pawwithdrawal from a radiant heat source (Hargreaves method: cutoff timeset at 18 sec.). Baseline latency ˜16 sec. Before any intrathecalinjections, the Naïve rats' responses to capsaicin injection into thehindpaw were determined to be: Tactile: 6 withdrawals/10 VFH probes, andThermal: 6 sec latency.

Intrathecal injections were made via a sacral approach, using a 30 gneedle, and delivering either SSP-SAP (modified Substance P-saporinconjugate), which eliminates most NK!-R-expressing spinal neurons(Mantyh et al.,) or its inactive congener, Blank-SAP (nonsense peptideconjugated to saporin).

Several weeks later, when the acute hyper-responsiveness due tocapsaicin had been abolished in SSP-SAP-treated animals (but notBlank-SAP-treated ones), Stimulus Triggered Activation of Pluripotency(STAP) stem cells (see FIG. 26) were injected into the same region ofthe lumbar spinal cord where SAP conjugates had been injected.

Responses to tactile and thermal stimulation after capsaicin injectionwere followed for another 5 weeks, at which time the rats wereanesthetized with pentobarbital (75 mg/kg i.p.) and cardio-perfused withcold saline, then 4% paraformaldehyde. Spinal cords were sectioned at 50um thickness and stained with anti-NK1-R and anti-neuron primaryantibodies: [Rabbit ant-NK-1R (lot#011M4819, Sigma-Aldrich St. Louis,Mo.) 1:5000 (2.3 μg/ml) and Mouse anti-NeuN (lot#LV1825845, MilliporeBillerica, Mass.) 1:500 (2 ug/ml), dissolved in PBS with 1% NDS and 0.3%Triton X-100], then washed extensively and incubated in the correlate 2°Abs [Donkey anti-Rabbit Alexa Fluor 555 (lot#819572) and Donkeyanti-Mouse Alexa Fluor 488 (lot#1113537) (Invitrogen, Grand Island,N.Y., USA), both 1:1000 (2 μg/ml) and were dissolved with 1% NDS and0.3% Triton X-100 in PBS] before viewing in a fluorescence microscope.

Total numbers of NK-1 and Neu-N immunopositive cell bodies per tissuesection were counted for superficial (I and II) and deep (III-V) laminaefrom each of the experimental groups (n=3/group). Results are expressedas mean percentages of surviving neurons in superficial and deep laminaein rats that received SSP-SAP (or vehicle) with or without stem celltreatment.

Mechanical hyperalgesia, indicated by the drop in paw withdrawalthreshold after capsaicin injection, is reduced in rats treated withintrathecal SSP-SAP (FIG. 27). Five weeks after spinal stem cells wereimplanted the capsaicin-induced hyperalgesia is restored. Stem cellimplant returned the hyperalgesic response of SSP-SAP-treated rats tothat of Naïve rats and of Blank-SAP-treated controls (FIG. 28). Thepotency of a specific antagonist of the NK1-R is increased in rats wherecapsaicin sensitivity has been restored by stem cell implants (FIG. 29).

SSP-SAP is highly effective in ablating NK1R-expressing neurons in thespinal cord and, thusly, of virtually abolishing the early hyperalgesicresponses to the capsaicin injected into the hind paw. Delivery of STAPstem cells restores the “normal” hyperalgesic tactile and thermalresponses to capsaicin in SSP-SAP treated rats. In rats that experiencedno change in hyperalgesic responsiveness due to the injection ofBlank-SAP, the delivery of STAP stem cells had no effect on theresponses to capsaicin. The normalization of the hyperalgesic responsesby STAP stem cells was accompanied by a return of NK1R-IR in the spinalcord. The potency of an antagonist of NK1R for inhibition of capsaicinhyperalgesia was enhanced 10-60 times in STAP stem cell restored ratsover its potency in Naïve rats or in rats that received Blank-SAP.Without wishing to be bound by theory, it is contemplated that thismight occur from a change in the affinity of the antagonist for the NK1Rinduced by the STAP stem cells or in a difference in the coupling ofNK1R into the overall scheme for hyperalgesic responses in the restoredrats.

Example 7 Described herein is a protocol with improved results increating pluripotent STAP stem cells from mature somatic cells, notdependent on the source of cells. The protocol has been revised toreflect improved techniques. This protocol utilizes a combination ofindividual stresses and approaches that are more effective in achievingthe desired end result; that is, creation of pluripotent STAP

Without wishing to be bound by theory, is contemplated herein that insome protocols described herein, ATP was utilized as an energy source toimprove the viability of the cells and spheres generated. The additionof ATP resulted in better sphere formation and was associated with amarked decrease in the pH of the solution to which the mature cells wereexposed. Further exploration of the utility of a low pH solutioncontaining ATP in generating. STAP cells indicates that while pH aloneresulted in the generation of STAP cells, the use of a low pH solutioncontaining ATP, dramatically increased the efficacy of this conversion.When this solution is used in combination with mechanical trituration ofmature cells, the results were even more profound. Consequently,described herein is a protocol which incorporates these findings toincrease the efficacy of generating STAP cells.

The described protocol is efficient for generating, STAP cells,regardless of the cell type being studied. In some embodiments,trituration of the mature cell suspension in the low pH. ATP enhancedsolution proceeds for a minimum of 30 minutes, e.g., until thesuspension can be easily triturated up and down the reduced borepipettes of the smallest orifices. First described is a protocol for usewhen starting with a suspension of cells, and then additional stepsnecessary when starting with a soft tissue are described.

A. Generating STAP Cells when Starting with a Suspension of MatureSomatic Cells:

A1. Make a low pH HBSS solution containing ACT as follows and then setaside for use in step A4. Make a stock solution of ATP, 200 mM, to addto HBSS by adding 110.22 mg of ATP powder (Adenosine 5′ TriphosphateDisodium Salt Hydrate—Sigma A2383) to each 1 mL of water (MilliQ water).The pH of this solution is about 3.0.

Place 5 mL of HBSS (with phenol red) [Life Technologies, 14170-161] intoa 15 mL tube. Place a clean pH sensor into the HBSS. Titrate in the ACTstock solution, drop by drop, into the HBSS until the desired pH of,e.g., 5.0 is obtained. Mix the solution regularly to ensure that themeasurement is accurate.

In some embodiments, the concentration of ATP in the resulting solutionof HBSS and ATP is from about 0.5 mg/cc to about 100 mg/cc. In someembodiments, the concentration of ATP in the resulting solution of HBSSand ATP is from about 0.5 mg/cc to about 20 mg/cc. In some embodiments,the concentration of ATP in the resulting solution of HBSS and ATP isfrom about 0.5 mg/cc to about 10 mg/cc. In some embodiments, theconcentration of ATP in the resulting solution of HBSS and ATP is fromabout 1.0 mg/cc to about 7 mg/cc. In some embodiments, the concentrationof ATP in the resulting solution of HBSS and ATP is from about 1.5 mg/ccto about 5 mg/cc. In some embodiments, the concentration of ATP in theresulting solution of HBSS and ATP is from about 1 mM to about 150 mM.In some embodiments, the concentration of ATP in the resulting solutionof HBSS and ATP is from about 1 mM to about 50 mM. In some embodiments,the concentration of ATP in the resulting solution of HBSS and ATP isfrom about 1 mM to about 15 mM. In some embodiments, the concentrationof ATP in the resulting solution of HBSS and ATP is from about 2.0 mM toabout 10 mM. In some embodiments, the concentration of ATP in theresulting solution of HBSS and ATP is from about 2.7 mM to about 9 mM.

In some embodiments, the concentration of ATP in the resulting solutionof HBSS and ATP is at least about 0.5 mg/cc. In some embodiments, theconcentration of ATP in the resulting solution of HBSS and ATP is atleast about 1.0 mg/cc. In some embodiments, the concentration of ATP inthe resulting solution of HBSS and ATP is at least about 1.5 mg/cc. Insome embodiments, the concentration of ATP in the resulting solution ofHBSS and ATP is at least about 1 mM. In some embodiments, theconcentration of ATP in the resulting solution of HBSS and ATP is atleast about 2.0 mM. In some embodiments, the concentration of ATP in theresulting solution of HBSS and ATP is at least about 2.7 mM.

In some embodiments, higher concentrations of ATP can be achieved bybuffering the solution at and/or around the desired pH. In someembodiments, the desired pH is at least 5.0. In some embodiments, thedesired pH is at least about 5.0. In some embodiments, the desired pH isabout 5.0. In some embodiments, the desired pH is at from about 4.0 toabout 6.5. In some embodiments, the desired pH is at from about 5.0 toabout 5.7.

A2. Add the live somatic cells to be treated, as a cell suspension to acentrifuge tube, and then centrifuge at 1200 rpm for 5 minutes. In someembodiments, 0.05% (Gibco: 25300-054) can be added to the tissue culturedish containing cells, for 3-5 minutes, to release adherent cells to beadded to the centrifuge tube.

A3. Aspirate the supernatant down to the cell pellet

A4. Resuspend the resulting pellet at a concentration of 1×106 cells/mlin the low pH, Hanks Balanced Saline Solution with ATP, (made above inStep 1A) in a 50 ml tube. In some embodiments, a volume of 2-3 ml of thecell suspension in a 50 ml tube can be used.

A5. Precoat a standard 9″ glass pipette with media (so the cells do notstick to the pipette—e.g., Fisher brand 9″ Disposable Pasteur Pipettes:13-678-20D). Triturate the cell suspension in and out of the pipette for5 minutes to dissociate cell aggregates and any associated debris. Thiscan be done with a fair amount of force.

A6. As a final step in the trituration process, make two fire polishedpipettes with very small orifices as follows: Heat the standard 9″ glasspipette over a Bunsen burner and then pull and stretch the distal(melting) end of the pipette, until the lumen collapses and the tipbreaks off, leaving a closed, pointed glass tip. Wait until the pipettecools, and then break off the closed distal tip until a very small lumenis now identifiable. Repeat this process with the second pipette, butbreak the tip off a little more proximally, creating a slightly largerdistal lumen. The larger lumen should be about 100-150 microns indiameter, while the other pipette should have a smaller lumen of about50-70 microns. Now triturate the cell suspension through the pipettewith the larger lumen for 10 minutes. Follow this with triturationthrough the pipette having the smaller lumen (50-70 microns) for anadditional 15 minutes. Continue to triturate the suspension until itpasses easily up and down the fire polished pipette of the smaller bore.Again, remember to precoat a each pipette with media. Also, duringtrituration, try to avoid aspirating air and creating bubbles or foam inthe cell suspension.

A7. Add normal HBSS (containing no ATP) to the suspension to a totalvolume of 20 ml, centrifuge at 1200 rpm for 5 minutes and then aspiratethe supernatant.

A8. Resuspend the resulting pellet in 5 ml of what we term “spheremedia” (DMEM/F12 with 1% Antibiotic and 2% B27 Gibco 12587-010) andplace at a concentration of 105 cells/ml, within a non-adherent tissueculture dish in the presence of the following supplements: b-FGF (20ng/ml), EGF (20 ng/ml), heparin (0.2%, Stem Cell Technologies 07980).LIF (1000 U) should be added if the cells are murine). In someembodiments, supplements such as bFGF, EGF and heparin may not benecessary. After the cells are placed in tissue culture dishes, theyshould be gently pipetted using a 5 ml pipette, twice/day for 2 minutes,for the first week, to discourage them from attaching to the bottom ofthe dishes. This is important to generate good sphere formation. Addsphere media containing the supplements described every other day. (Add1 ml/day to a 10 cm culture dish, or 0.5 ml/day to a 6 cm dish.)

B. Generating ST AP Cells when Starting with Soft Tissues that ContainMany RBCs.

B1. Place the excised, washed sterile organ tissue into an 60 mm petridish containing 50-500 μl of collagenase, depending on the size of thetissue. Add a sufficient volume of the collagenase to wet the entiretissue. Different types of collagenase or other enzymes are better fordigestion of different organ tissues. (The spleen may not need to beexposed to any digestive enzymes.)

B2. Mince and scrape the tissue for 10 minutes using scalpels andscissors to increase surface area that is exposed to the collagenase,until the tissue appears to become gelatinous in consistency.

It is specifically contemplated herein that in this embodiment of themethod, or any embodiment of the method described herein, that thescraping of the tissue can be performed with a flat edged blade and/orsurface, e.g., a number 11 scalpel as opposed to a curved surgicalblade. Alternatively, in any embodiment described herein, application ofhigh frequency sound waves can be substituted for scraping and/orcombined (either simultaneously or sequentially) in order to disrupt thetissue. High frequency sound waves can, e.g., disrupt membranes, punchholes in tissue and/or membranes, and/or cause membrane leakiness. Highfrequency sound waves are also amenable being scaled up. One of skill inthe art is familiar with methods for applied high frequency sound wavesto tissues, e.g., commercial sonicators are available (e.g. The QsonicaQ55 Sonicator, Cat. No. UX-04712-52 available from Cole-Palmer; VernonHills, Ill.).

B3. Add additional collagenase to the dish to make the total volume=0.5ml, and place in a incubator/shaker for 30 minutes at 37° C. at 90 rpm.

B4. Add 1.5 ml of the low pH HBSS/ATP solution to the dish (yielding atotal volume of 2.0 ml) and then aspirate the entire contents via a 5 mlpipette and place into a 50 ml tube.

B5. Now proceed to triturate as previously described above (step A4-5)when starting with mature somatic cells.

B6. After trituration is completed (through step A5 when using a culturedish of mature somatic cells), add 3 ml of HBSS, yielding a volume of 5ml, to the 15 ml tube and then slowly add 5 ml of Lympholyte to thebottom of the tube to create a good bilayer. The solution should beadded as described to create a bilayer and avoid mixing of the twosolutions.

B7. Centrifuge this tube at 1000 g for 10 min Rotate the tube 180° andrecentrifuge at 1000 g for an additional 10 min. This will cause theerythrocytes to form a pellet at the bottom of the tube.

B8. Using a standard 9″ glass pipette, aspirate the cell suspensionslayer between HBSS and Lympholyte and place in a new 50 ml tube.

B9. Add HBSS to the suspension to a total volume of 20 ml of HBSS andthen thoroughly mix the suspension by pipetting via a 5 ml pipette for 1minute.

B10. Centrifuge the solution at 1,200 rpm for 5 minutes and aspirate thesupernatant.

B11. For the next steps see A6-8.

1. A method to generate a pluripotent cell, comprising: a. Isolating aninitial cell from a solution; b. Resuspending a cell resulting from stepa in a Balanced Saline Solution (BSS); c. Resuspending the cellresulting from step b in BSS having a pH of about 5.0 to about 6.0; d.Triturating the cell suspension resulting from step c; e. Isolating acell from the suspension resulting from step d; f. Incubating the cellsat about their natural in vivo temperature; g Isolating a cell from thesuspension resulting from step f; and h. Resuspending the cell pelletresulting from step g in media. 2-5. (canceled)
 6. The method of claim1, wherein the trituration of step d comprises triturating the cellsthrough apertures or lumens. 7-28. (canceled)
 29. The method of claim 1,wherein step f comprises incubating the cells about 37° C. for about 25minutes. 30-31. (canceled)
 32. The method of claim 1, wherein the mediaof step i is Sphere Media comprising DMEM/F12, about 1% antibiotic,about 2% B27, and optionally, one or more growth factors. 33-60.(canceled)
 61. A method to generate a pluripotent cell, comprising: a.Isolating an initial cell from a solution; b. Resuspending a cellresulting from step a in a solution of a Balanced Saline Solution (BSS)and ATP having a pH of from about 4.0 to about 6.5; c. Triturating thecell suspension resulting from step b; d. Resuspending the cellresulting from step c in BSS; e. Isolating a cell from the suspensionresulting from step d; and f Resuspending the cell pellet resulting fromstep g in media.
 62. A method to generate a pluripotent cell,comprising: a. Isolating an initial cell from a solution; b.Resuspending a cell resulting from step a in a solution of a BalancedSaline Solution (BSS) and ATP having a pH of from about 4.0 to about6.5; c. Isolating a cell from the suspension resulting from step b; d.Resuspending the cell resulting from step c in a solution of BSS and ATPhaving a pH of from about 4.0 to about 6.5 to the cell suspension; e.Triturating the cell suspension resulting from step d; f. Resuspendingthe cell resulting from step e in BSS; g. Isolating a cell from thesuspension resulting from step f; and h. Resuspending the cell pelletresulting from step g in media.
 63. The method of claim 61, wherein theATP is present in the solution of BSS and ATP at a concentration of fromabout 1.5 to about 5 mg/cc.
 64. The method of claim 63, wherein the ATPis present in the solution of BSS and ATP at a concentration of fromabout 2.7 mM to about 9 mM. 65-74. (canceled)
 75. The method of claim61, wherein the trituration comprises triturating the cells throughapertures or lumens. 76-97. (canceled)
 98. The method of claim 61,wherein the media is Sphere Media comprising DMEM/F12, about 1%antibiotic, about 2% B27, and optionally, one or more growth factors.99-152. (canceled)
 153. The method of claim 1, wherein the initial cellis a somatic cell, a stem cell, a progenitor cell or an embryonic cell.154-156. (canceled)
 157. The method of claim 1, wherein selecting thecell exhibiting pluripotency comprises selecting a cell expressing astem cell marker selected from the group consisting of Oct4, Nanog,E-cadherin, and SSEA4. 158-174. (canceled)
 175. The method of claim 62,wherein the ATP is present in the solution of BSS and ATP at aconcentration of from about 1.5 to about 5 mg/cc.
 176. The method ofclaim 175, wherein the ATP is present in the solution of BSS and ATP ata concentration of from about 2.7 mM to about 9 mM.
 177. The method ofclaim 62, wherein the trituration comprises triturating the cellsthrough apertures or lumens.
 178. The method of claim 62, wherein thetotal time of trituration is about 30 minutes.
 179. The method of claim62, wherein the media is Sphere Media comprising DMEM/F12, about 1%antibiotic, about 2% B27, and optionally, one or more growth factors.